CN115925957A

CN115925957A - Specific sites for modifying antibodies to make immunoconjugates

Info

Publication number: CN115925957A
Application number: CN202210867143.7A
Authority: CN
Inventors: B·H·盖尔斯坦格尔; W·欧; 宇野哲郎
Original assignee: IRM LLC
Current assignee: Novartis International Pharmaceutical Ltd; Novartis AG; Novartis Pharma AG
Priority date: 2013-02-08
Filing date: 2014-02-07
Publication date: 2023-04-07
Also published as: EP2953976A2; KR20220133313A; HK1213580A1; SG10201706468RA; BR112015018899A2; KR102626525B1; EP3929217A3; AU2014214751A1; WO2014124316A3; CN105143271A; ES2874493T3; CA2900755C; KR102447350B1; IL240406A0; AU2014214751B2; EP2953976B1; US20230270876A1; MX2015010146A; HUE054443T2; US11596695B2

Abstract

The present invention provides for modifying specific sites of an antibody or antibody fragment by replacing at least one natural amino acid in the constant region of a parent antibody or antibody fragment with a cysteine, which can be used as an attachment site for a payload or a linker-payload combination.

Description

Specific sites for modifying antibodies to make immunoconjugates

The present application is a divisional application of chinese patent application 201480017839.4, the filing date of the original application being 2/7/2014, entitled "specific site for modifying an antibody to prepare an immunoconjugate".

Technical Field

Due to the importance of antibodies for a variety of therapeutic applications, robust methods are needed to selectively modify antibodies to introduce improved properties or additional functions. The present invention provides for the attachment of moieties to antibodies at specific sites for the preparation of modified antibodies, such as for the preparation of antibody-drug conjugates (ADCs). Selective conjugation sites are localized to the constant region of the antibody and thus are used for a variety of antibodies.

Background

The value of methods for modifying antibodies is well known, and many methods have been developed for conjugating antibodies to attach various "payload" moieties. Many of these methods rely on the natural presence of specific reactive amino acid residues on the antibody, such as lysine and cysteine (which can be used to attach the payload). However, relying on natural amino acids is not always desirable, as the position and amount of payload attached depends on the number and position of these reactive amino acids: too many or too few of such residues make it difficult to effectively control the loading of the payload on the antibody. Furthermore, the placement of the reactive amino acids may make it difficult to obtain complete conjugation, resulting in heterogeneous products during conjugation. Heterogeneity of pharmaceutical active ingredients, for example, is generally undesirable as it exacerbates unpredictability of administering drugs to a heterogeneous population of subjects: it is more preferred to administer homogeneous products and it is much more difficult to fully characterize and predict the behavior of heterogeneous products. Cytotoxic drugs are site-specifically conjugated to antibodies, e.g., via engineered cysteine residues, to produce homogeneous immunoconjugates that exhibit improved therapeutic indices (Junutula et al, (2008) Nat Biotechnol.26(8):925-932))。

Antibodies have been engineered to add certain residues, like cysteines, at specific positions where these residues can be used for conjugation (Lyons et al, (1990) Protein eng., 3.

Because the engineered cysteines in antibodies expressed in mammalian cells are modified during their biosynthesis by disulfide bonds with Glutathione (GSH) and/or cysteine (Chen et al (2009) mAbs 1, 6,563-571), the modified cysteines in the originally expressed antibody drug conjugates do not react with thiol-reactive reagents. Activation of the engineered cysteine requires reduction of GSH and/or cysteine adducts (which typically results in reduction of all interchain disulfide bonds of the antibody), followed by re-oxidation and reformation of the native interchain disulfide bonds prior to conjugation (Junutula et al, (2008) nat. Biotechnol.26 (8): 925-32). Some sites where cysteines have been inserted interfere with the re-oxidation process, followed by the generation of unwanted non-homogeneous conjugation products. It is therefore important to identify sites on the antibody where the introduced cysteine does not interfere with the reoxidation process prior to conjugation with a thiol-reactive reagent such as a maleimide or bromo, chloro or iodoacetamide group.

Conjugation of cysteine residues to bromoacetamide, iodoacetamide, or chloroacetamide results in the formation of stable thioether linkages. (Alley et al (2008) bioconjugate Chem.19 (3): 759-65). However, the chemistry is not as efficient as the maleimide conjugation chemistry. Since the formation of this thiol-maleimide linkage is a popular and highly efficient method to be used when attaching a payload or linker to a cysteine, there is a need to identify cysteine substitution sites on antibodies where maleimide linkage can be used. More importantly, site-specifically conjugated immunoconjugates can exhibit improved therapeutic indices, and thus there remains a need to identify specific privileged sites for cysteine substitutions in antibodies that enable the conjugation of payloads onto antibodies to form efficiently and provide conjugates with high stability. The present invention provides such privileged cysteine substitution sites (which result in improved antibodies for conjugation purposes) and immunoconjugates comprising such improved antibodies.

Summary of The Invention

The present invention provides specific sites in the constant region of an antibody or antibody fragment at which cysteine ("Cys") substitutions of the natural amino acids on the parent antibody or antibody fragment can be made to provide Cys residues for attachment of chemical moieties (e.g., payload/drug moieties) to form immunoconjugates with good potency and stability. The invention also provides engineered antibodies or antibody fragments having one or more Cys residues in one or more of these specific sites, and immunoconjugates prepared from the engineered antibodies or antibody fragments.

Methods for inserting Cys at a specific position in an antibody are known in the art, see, e.g., WO 2011/005481. However, the present invention discloses specific sites in the constant region of an antibody or antibody fragment, wherein the replacement of one or more natural amino acids of the parent antibody or antibody fragment with Cys provides one or more of the following advantages: good reactivity to promote efficient immunoconjugates; reduced propensity to lose payload when using a Cys-maleimide conjugated linker; a reduced tendency to form unwanted disulfide bonds between antibodies, such as cross-linking or formation of non-native intramolecular disulfide bonds; and low hydrophobicity of the resulting ADC.

The particular privileged site for the Cys substitution of a natural amino acid in the constant region of a parent antibody or antibody fragment is selected to provide efficient conjugation while minimizing unwanted effects. First, the specific site for modification is selected so that replacing the natural amino acid of the parent antibody or antibody fragment in one or more selected positions provides an antibody or antibody fragment that is easily conjugated on a new cysteine. The specific positions are selected to be sufficiently surface accessible to allow the thiol group of the cysteine residue to react with an electrophile in aqueous solution. Identifying suitable sites for Cys substitution of a natural amino acid of a parent antibody or antibody fragment involves analyzing the surface exposure of the natural amino acid based on crystal structure data. Because the sites described herein are sufficiently accessible and reactive, they can be used to form immunoconjugates by chemical actions well known in the art for modifying naturally occurring cysteine residues. Conjugation of replacement Cys residues thus uses conventional methods.

When a thiol-maleimide moiety is used for conjugation, the selected modification site may show a low propensity for reverse conjugation. Thiol-maleimide conjugation reactions are often highly selective and extremely efficient, and can be used to attach a payload to a thiol group of a cysteine residue of a protein or elsewhere as a linker in a linkage between a protein and a payload. For example, a maleimide may be attached to a protein (e.g., an antibody or antibody fragment), and a payload having an attached thiol group may be added to the maleimide to form a conjugate:

thus, in this conjugation step, the protein (e.g., antibody or antibody fragment) may be monocyclic or bicyclic; and the other representing the payload. The immunoconjugate stability information herein specifically relates to conjugation of a substituted cysteine by reaction with a maleimide group. In some embodiments, the sulfhydryl group is from a cysteine on the protein (e.g., an antibody or antibody fragment), so that a bicyclic ring represents the protein and a monocyclic ring represents the payload.

Although thiol-maleimide reactions are often used to prepare conjugates, reversal of the conjugation step as described below can result in loss of payload or confusion of payload with other thiol-containing species:

Some sites for cysteine substitution have been reported to provide more stable maleimide conjugates than others, presumably because the local chemical environment at certain points on the antibody surface around the new cysteine can promote hydrolysis of the succinimide ring and thus prevent reversal of the thiol-maleimide linkage in the immunoconjugate (Shen et al (2012), nat. Biotechnol.30 (2): 184-9). Identifying favorable sites for meeting this criteria involves inserting Cys in place of many natural amino acids with appropriate surface exposure, preparing immunoconjugates containing thiol-maleimide linkages, and evaluating the stability of the immunoconjugate to remove sites where the stability of the conjugate is reduced by the local microenvironment around the destabilization site. Based on this, the chemistry that can be used to attach the linker and payload to the replacement Cys residue is not limited by the stability issues associated with the reversibility of the thiol-maleimide conjugate discussed above. A number of methods can be used to form conjugates on cysteines, including maleimide conjugation. The sites described herein for cysteine substitution facilitate the stability of the antibody conjugate product when one of the most common conjugation methods is used, making these sites advantageous for antibody engineering, as the modified antibodies can be used in well-known and highly efficient maleimide conjugation methods. Site selection based on this criterion is illustrated by the data given in table 22 and example 9.

The selected site can be located so as to minimize unwanted disulfide formation (which may interfere with the formation of homogeneous conjugates). When antibodies or antibody fragments containing engineered cysteines are produced in mammalian cells, the Cys residues are usually present as disulfides with free Cys amino acids or glutathione (Chen et al, (2009) mAbs 16, 353-571). To release the Cys residue for conjugation with a thiol-reactive group, the antibody or antibody fragment needs to be reduced, breaking all disulfide bonds. The antibody or antibody fragment is then reoxidized under conditions that promote the formation of the native disulfide (which stabilizes the antibody or antibody fragment). After reoxidation, cysteine residues that are too significantly exposed on the surface of an antibody or antibody fragment may form disulfides by reacting with Cys on another antibody or antibody fragment ("inter-antibody disulfides") or by forming unwanted intra-antibody disulfides. It has been found that cysteine residues placed at specific positions described herein are suitably easy to use for effective conjugation, but are sufficiently shielded or appropriately positioned to reduce or eliminate inter-and intra-antibody disulfide bond formation that would otherwise occur during the reduction/reoxidation process typically required when expressing cys-modified antibodies. Similarly, upon reoxidation, some sites were found to produce non-homogeneous conjugation products, which appears to be due to the engineered new Cys residues being located into the protein, and the specific sites identified herein are some where such heterogeneity is minimized.

Preferably, the drug payload is conjugated at a site where the payload is sequestered from solvent interactions and attachment may increase the hydrophobicity of the antibody after drug attachment, as it is generally believed that decreasing the hydrophobicity of the protein drug is beneficial because it may reduce aggregation and clearance from circulation. When 4, 6 or 8 drugs are attached to each antibody, or when a particularly hydrophobic payload is used, it may be particularly beneficial to select an attachment site that results in the least change in hydrophobicity.

Using these and additional methods described in the examples herein to assess sites for Cys integration results in selection of preferred sites for Cys integration for engineering antibodies or antibody fragments to introduce Cys as a site for conjugation, particularly for preparation of ADCs. Additional details regarding the selection of specific sites for the natural amino acids of the Cys replacement antibody are provided herein.

The cysteine substitution site is located in the constant region of the antibody or antibody fragment and is identified herein using standard numbering conventions. However, it is well known that portions or fragments of antibodies, rather than complete full length antibodies, can be used for many purposes, and as such antibodies can be modified in a variety of ways that affect the numbering of sites in the constant region, even if they do not substantially affect the function of the constant region. For example, it has been shown that an S6 tag (short peptide) is inserted into the loop region of an antibody to allow the activity of the antibody to be retained, even though it may alter the numbering of many sites in the antibody. Thus, while the preferred cysteine substitution sites described herein are identified by a standard numbering system based on the numbering of intact antibodies, the invention encompasses antibody fragments or corresponding sites in antibodies containing other modifications, such as peptide tag insertions. Those fragments or corresponding sites in the modified antibody are thus preferred sites for cysteine substitutions in the fragment or modified antibody, and reference to a cysteine substitution site by number includes a corresponding site in the modified antibody or antibody fragment that retains the function of the relevant portion of the full-length antibody.

The corresponding sites in an antibody fragment or modified antibody can be readily identified by aligning a segment of the antibody fragment or modified antibody with a full-length antibody to identify a site in the antibody fragment or modified antibody that matches one of the preferred cysteine substitution sites of the present invention. The alignment may be based on a segment that is long enough to ensure that the segment matches the correct portion of the full-length antibody, such as a segment of at least 20 amino acid residues, or at least 50 residues, or at least 100 residues, or at least 150 residues. The alignment may also take into account other modifications that have been engineered into the antibody fragment or modified antibody, thus allowing sequence differences due to engineered point mutations, particularly conservative substitutions, in the segments used for alignment. Thus, for example, an Fc domain may be excised from an antibody and will contain amino acid residues corresponding to the cysteine substitution sites described herein, despite the numbering differences: it is also contemplated that a site in the Fc domain corresponding to a cysteine substitution site of the invention is a favorable site for cysteine substitution in the Fc domain and is included within the scope of the invention.

In one embodiment, the invention provides an immunoconjugate of formula (I):

Wherein Ab represents an antibody or antibody fragment comprising at least one cysteine residue at one of the preferred cysteine substitution sites described herein;

LU is a joint unit as described herein;

x is a payload or drug moiety;

and n is an integer from 1-16.

Typically, in the compound of formula (I), LU is attached to a cysteine at one of the cysteine substitution sites described herein, X is a drug moiety, such as an anti-cancer drug, and n is 2 to 8 when Ab is an antibody, or n may be 1 to 8 when Ab is an antibody fragment.

In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at positions 121, 124, 152, 171, 174, 258, 292, 333, 360 and 375 selected from the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at positions 107, 108, 142, 145, 159, 161, and 165 of a light chain selected from said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

In one embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at positions 143, 147, 159, 163 and 168 of a light chain selected from the antibody or antibody fragment, wherein the positions are numbered according to the Kabat system, and wherein the light chain is a human lambda light chain.

In one embodiment, the invention provides a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine at a position described herein. The sites for cysteine substitution are located in the constant region of the antibody and thus are applicable to a variety of antibodies, and the sites are selected to provide stable and homogeneous conjugates. The modified antibody or fragment may have two or more cysteine substitutions, and these substitutions may be used in combination with other antibody modification and conjugation methods as described herein.

In one embodiment, the invention provides pharmaceutical compositions comprising the immunoconjugates disclosed above, and methods of using the immunoconjugates.

In one embodiment, the invention provides a nucleic acid of this, encoding a modified antibody or antibody fragment described herein having at least one cysteine substitution at a position described herein. The invention also provides host cells comprising these nucleic acids and methods of using the nucleic acids or host cells to express and produce the antibodies or fragments described herein.

In one embodiment, the present invention provides a method to select amino acids of an antibody suitable for substitution by cysteine to provide good conjugation sites, comprising:

(1) Identifying amino acids in the antibody constant region that have suitable surface exposures to provide a set of initial candidate sites;

(2) For each of the initial candidate sites, expressing an antibody in which the natural amino acid at that site is replaced with cysteine;

(3) For each expressed antibody, determining whether the expressed protein is substantially homogeneous after reduction and reoxidation to provide a functional antibody having a free cysteine at the initial candidate site,

(4) For each protein expressed that is substantially homogeneous and functional, conjugating a cysteine at the initial candidate site to a maleimide moiety and determining whether the thiol-maleimide linkage is stable at that site;

(5) Those sites for which the expressed antibody is substantially non-homogeneous and non-functional, and those sites in which the thiol-maleimide linkage is labile, are removed from the set of initial candidate sites to provide a set of favorable sites for cysteine substitution.

Optionally, the method further comprises determining a melting temperature for each favorable cysteine substitution site conjugate and excluding any such sites from the set, wherein the cysteine substitution and conjugation results in a melting temperature that is 5 ℃ or greater different from the melting temperature of the native antibody.

In one embodiment, the invention provides a method to produce an immunoconjugate comprising attaching a Linker Unit (LU) or linker unit-payload combination (-LU-X) to a cysteine residue in an antibody or antibody fragment, wherein the cysteine is positioned at a cysteine substitution site selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 360, and 375 of a heavy chain of the antibody or antibody fragment and a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system.

Other aspects and embodiments of the invention are described in more detail herein.

1. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at a position selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

2. The immunoconjugate of embodiment 1, wherein the substitution of one or more amino acids with cysteine is selected from positions 121, 124, 152, 258, 334, 360, and 392.

3. The immunoconjugate of

embodiment

1 or 2, wherein said antibody or antibody fragment comprises a sequence selected from

SEQ ID NOs

4, 5, 10, 17, 18, 29, 35, 42, 43, 48, 50, 54, 290, 291, 292, 293, 294, and 295.

4. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at a position selected from positions 107, 108, 142, 145, 159, 161, and 165 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

5. The immunoconjugate of embodiment 4, wherein the substitution of one or more amino acids with cysteine is selected from positions 145 or 165.

6. The immunoconjugate of embodiment 4, wherein said antibody or antibody fragment comprises a sequence selected from SEQ ID NOs:61, 62, 69, 71, 75, 76, and 77.

7. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at positions selected from positions 143, 147, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

8. The immunoconjugate of embodiment 7, wherein said antibody or antibody fragment comprises a sequence selected from SEQ ID NOs:92, 94, 96, 97, and 98.

9. The immunoconjugate of

embodiment

1, 2 or 3, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with cysteine on its constant region at a position selected from positions 107, 108, 142, 145, 159, 161, and 165 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

10. The immunoconjugate of

embodiment

1, 2 or 3, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with cysteine on its constant region at positions 143, 147, 159, 163, and 168 of a light chain selected from said antibody or antibody fragment, wherein said light chain positions are numbered according to the Kabat system, and wherein said light chain is a human kappa light chain.

11. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of cysteine substitutions to two or more amino acids at positions 152 and 375, or at positions 327 and 375, on a heavy chain constant region, wherein said positions are numbered according to the EU system.

12. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises on its constant region a combination of substitutions of two or more amino acids by cysteines at position 107 of a light chain and 360 of a heavy chain, wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

13. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from

positions

117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400, or 422 of the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

14. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from

positions

107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of the light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

15. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on a light chain constant region of said antibody or antibody fragment selected from positions 143, 145, 147, 156, 159, 163, and 168, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

16. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of cysteine substitutions to two or more amino acids on its constant region, wherein said combination comprises substitutions at position 375 of an antibody heavy chain and position 165 of an antibody light chain, or at position 334 of an antibody heavy chain and position 165 of an antibody light chain, and wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

17. The immunoconjugate of any one of embodiments 11, 12 and 16, wherein the drug-to-antibody ratio is about 4.

18. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of cysteine to three or more amino acid substitutions on its constant region, wherein said combination comprises a substitution selected from

a. Positions 375 and 392 of the antibody heavy chain and position 165 of the antibody light chain,

b. positions 334 and 375 of the antibody heavy chain and position 165 of the antibody light chain, and

c. substitutions at positions 334 and 392 of the antibody heavy chain and position 165 of the antibody light chain,

and wherein the light chain is a kappa chain, and wherein the positions are numbered according to the EU system.

19. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of cysteine to three or more amino acid substitutions on its constant region, wherein said combination comprises a substitution selected from

a. Positions 152, 375 and 392 of the heavy chain of the antibody,

b. positions 152, 334 and 375 of the heavy chain of the antibody, and

c. substitutions at positions 152, 334 and 392 of the antibody heavy chain,

and wherein the positions are numbered according to the EU system.

20. The immunoconjugate of embodiment 18 or 19, wherein the drug-to-antibody ratio is about 6.

21. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of cysteine substitutions of four or more amino acids on its constant region, wherein said combination comprises substitutions at positions 334, 375, and 392 of an antibody heavy chain and position 165 of an antibody light chain, or positions 333, 375, and 392 of an antibody heavy chain and position 165 of an antibody light chain, and wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

22. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of four or more amino acids with cysteine on its constant region, wherein said combination comprises substitutions at positions 152, 334, 375, and 392 of an antibody heavy chain, or at positions 152, 333, 375, and 392 of an antibody heavy chain, and wherein said positions are numbered according to the EU system.

23. The immunoconjugate of embodiment 21 or 22, wherein the drug-to-antibody ratio is about 8.

24. The immunoconjugate of any one of embodiments 1-23, further comprising a drug moiety.

25. The immunoconjugate of embodiment 24, wherein the drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker.

26. The immunoconjugate of embodiment 25, wherein said drug moiety is linked to said sulfur of said cysteine through a cleavable or non-cleavable linker.

27. The immunoconjugate of embodiment 25, wherein said drug moiety is linked to said sulfur of said cysteine through a non-cleavable linker.

28. The immunoconjugate of embodiment 25, wherein said immunoconjugate comprises a thiol-maleimide linkage.

29. The immunoconjugate of embodiment 25, wherein said immunoconjugate comprises-S-CH ₂ -C (= O) -linkage or disulfide bond.

30. The immunoconjugate of any one of embodiments 25-29, wherein said drug moiety is a cytotoxic agent.

31. The immunoconjugate of embodiment 30, wherein the drug moiety is selected from the group consisting of taxanes, DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin (auristatin) E, auristatin F, and maytansinoids (maytansinoids).

32. The immunoconjugate of any one of embodiments 1-31, wherein said antibody is a monoclonal antibody.

33. The immunoconjugate of any one of embodiments 1-31, wherein said antibody is a chimeric antibody.

34. The immunoconjugate of embodiment 31, wherein said antibody is a humanized or fully human antibody.

35. The immunoconjugate of embodiment 31, wherein said antibody is a bispecific or multispecific antibody.

36. The immunoconjugate of any one of embodiments 1-32, wherein said antibody or antibody fragment specifically binds to a cell surface marker characteristic of a tumor.

37. A pharmaceutical composition comprising the immunoconjugate of any one of embodiments 1-36.

38. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine in its constant region selected from

position

117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 or 422 of the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

39. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine on its constant region at

positions

107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199 and 203 of a light chain selected from said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

40. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine at positions 143, 145, 147, 156, 159, 163, 168 on a constant region of a light chain selected from said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

41. The modified antibody or antibody fragment of embodiment 38, wherein the substitution is at least one cysteine selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 360 and 375 of the heavy chain, and wherein the positions are numbered according to the EU system.

42. The modified antibody or antibody fragment of embodiment 39, wherein said substitutions are two to six cysteines, wherein said cysteines are at positions selected from 121, 124, 152, 171, 174, 258, 292, 333, 360, and 375 of the heavy chain, and wherein said positions are numbered according to the EU system.

43. The modified antibody or antibody fragment of embodiment 39, wherein said substitution is at least one cysteine selected from positions 107, 108, 142, 145, 159, 161, and 165 of the light chain, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

44. The modified antibody or antibody fragment of embodiment 40, wherein said substitutions are two to six cysteines, wherein said cysteines are at positions selected from the group consisting of 107, 108, 142, 145, 159, 161, and 165 of the light chain, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

45. The modified antibody or antibody fragment of embodiment 40, wherein the substitution is at least one cysteine selected from positions 143, 147, 159, 163 and 168 of the light chain, wherein said positions are numbered according to the Kabat system, and wherein the light chain is a human λ light chain.

46. The modified antibody or antibody fragment of embodiment 40, wherein the substitution is two to six cysteines, wherein the cysteines are at positions selected from 143, 147, 159, 163, and 168 of the light chain, wherein the positions are numbered according to the Kabat system, and wherein the light chain is a human λ light chain.

47. The modified antibody or antibody fragment of any one of embodiments 11, 12, 14-22, 38-47, further attached to a drug moiety, and wherein said drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker.

48. The modified antibody or antibody fragment of embodiment 47, wherein said drug moiety is attached to the sulfur of said cysteine through a linker unit.

49. The modified antibody or antibody fragment of any one of embodiments 38-48, further comprising at least one Pcl or unnatural amino acid substitution or peptide tag for enzyme-mediated conjugation and/or combinations thereof.

50. A nucleic acid encoding the modified antibody or antibody fragment of any one of embodiments 38-49.

51. A host cell comprising the nucleic acid of embodiment 50.

52. A method of producing a modified antibody or antibody fragment comprising incubating the host cell of embodiment 49 under suitable conditions for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment.

53. A method of selecting amino acids of an antibody suitable for substitution by cysteine to provide suitable conjugation sites, comprising:

(4) For each protein expressed that is substantially homogeneous and functional, conjugating a cysteine at the initial candidate site to a maleimide moiety and determining whether the thiol-maleimide linkage is destabilized at that site;

(5) Those sites for which the expressed antibody is substantially non-homogeneous and non-functional, and those sites in which the thiol-maleimide linkage is destabilized, are removed from the set of initial candidate sites to provide a set of favored sites for cysteine substitutions.

54. The method of embodiment 53, further comprising determining a melting temperature for each favorable cysteine substitution site conjugate and excluding from the set any sites where the cysteine substitution and conjugation results in a melting temperature that is 5 ℃ or greater different from the melting temperature of the parent antibody.

55. The method of embodiment 53 or 54, further comprising producing an antibody or antibody fragment comprising a cysteine at the identified one or more substitution sites.

56. A method of producing an immunoconjugate, comprising attaching a Linker Unit (LU) or linker unit-payload combination (-LU-X) to a cysteine residue in an antibody or antibody fragment, wherein the cysteine is positioned at a cysteine substitution site selected from 121, 124, 152, 171, 174, 258, 292, 333, 360, and 375 of a heavy chain of the antibody or antibody fragment and 107, 108, 142, 145, 159, 161, and 165 of a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system.

57. The method of embodiment 56, wherein the immunoconjugate is of formula (I):

LU is a joint unit as described herein;

x is a payload or drug moiety;

and n is an integer from 1-16.

Definition of

The term "amino acid" refers to typical, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics, which function in a manner similar to typical amino acids. Typical amino acids are those produced by proteins encoded by the genetic code and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and selenocysteine, pyrrolysine, and the analogs pyrroline-carboxy-lysine. Amino acid analogs refer to compounds that have the same basic chemical structure as a typical amino acid, i.e., an alpha-carbon that binds a hydrogen, a carboxyl group, an amino group, and an R group, such as citrulline, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as typical amino acids.

Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a typical amino acid. As used herein, the term "unnatural amino acid" is intended to represent an amino acid structure that cannot be biosynthetically produced in any organism using unmodified or modified genes (whether identical or different) of any organism. In addition, such "unnatural amino acids" typically require the incorporation of modified tRNAs and modified tRNA synthetases (RSs) into proteins. The tRNA/RS pair preferentially incorporates unnatural amino acids over typical amino acids. Such orthogonal tRNA/RS pairs are generated by a selection method developed by Schultz et al (see, e.g., liu et al, (2010) Annu. Rev. Biochem.79: 413-444) or similar methods. The term "unnatural amino acid" does not include naturally occurring 22 ^nd The protein-produced amino acid pyrrolysine (Pyl) and its demethylated analog pyrroline-carboxy-lysine (Pcl) because the incorporation of both residues into the protein is mediated by an unmodified, naturally occurring pyrrollysyl-tRNA/tRNA synthetase pair and because Pyl and Pcl are produced biosynthetically (see, e.g., ou et al, (2011) Proc. Natl. Acad. Sci. USA,108, 10437-10442 Celliti et al, (2011) nat. Chem. Biol.27;7 (8): 528-30). See also U.S. provisional application 61/76236, incorporated by reference, site-specific amino acid residues in antibody light and heavy chains may be substituted with Pcl.

The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly and in a specific manner. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains (also referred to as "antibody heavy chains") and two light (L) chains (also referred to as "antibody light chains") interconnected by disulfide bonds. Each heavy chain bagHeavy chain-containing variable region (abbreviated herein as V) _H ) And a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V) _L ) And a light chain constant region. The light chain constant region comprises a domain, C _L 。V _H And V _L Regions may be further subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each V _H And V _L Consists of three CDRs and four FRs, and is arranged from an amino terminal to a carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention). The antibody can be of any isotype/type (e.g., igG, igE, igM, igD, igA, and IgY), or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2).

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used for functionality. In this respect, it will be understood that (V) _L ) Chain sum weight (V) _H ) The variable domains of the chain portions all determine antigen recognition and specificity. In contrast, light chain (C) _L ) And of the heavy chain (CH 1, CH2 or CH 3) confers important biological properties such as secretion, transplacental mobility, fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become further from the antigen binding site or amino terminus of the antibody. The N-terminus is a variable region and the C-terminus is a constant region; CH3 and C _L The domains actually comprise the carboxy-terminal domains of the heavy and light chains, respectively.

The term "antibody fragment" as used herein refers to an antigen-binding fragment of an antibody Or a non-antigen binding fragment of an antibody (e.g., fc). As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, single chain Fvs (scFv), disulfide linked Fvs (sdFv), fab fragments, F (ab') fragments, fragments consisting of V _L 、V _H 、C _L And a CH1 domain; f (ab) ₂ A fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; from V _H And a CH1 domain; v on single arm of antibody _L And V _H (iii) a domain consisting of an Fv fragment; dAb fragments (Ward et al, nature 341, 544-546, 1989) derived from V _H Domain composition; and isolated Complementarity Determining Regions (CDRs), or other epitope-binding fragments of antibodies.

Furthermore, despite the two domains V of the Fv fragment _L And V _H Encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that allow them to be prepared as a single protein chain, where V _L And V _H The regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g., bird et al, science 242, 423-426,1988; and Huston et al, proc.Natl.Acad.Sci.85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment". These antigen binding fragments can be obtained using conventional techniques known to those skilled in the art and screened for usefulness in the same manner as intact antibodies.

Antigen binding fragments may also be incorporated into single domain antibodies, macroantibodies (maxibodes), miniantibodies (minibodies), nanobodies (nanobodies), intrabodies (intrabodies), diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies), v-NAR and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology 23, 1126-1136, 2005). Antigen-binding fragments can be grafted onto a polypeptide-based scaffold, such as fibronectin type III (Fn 3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide mono-antibodies).

Antigen binding fragments can be incorporated into a chimeric antibody comprising a pair of tandem Fv segments (V) _H -CH1-V _H -CH 1) which, together with a complementary light chain polypeptide, form a pair of antigen binding regions (Zapata et al, protein Eng.8:1057-1062,1995; and U.S. Pat. No. 5,641,870).

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides comprising antibodies and antibody fragments having substantially the same amino acid sequence or from the same genetic source. The term also includes preparations of antibody molecules that are composed of a combination of single molecules. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

As used herein, the term "human antibody" includes antibodies having variable regions in which the two framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibodies containing consensus framework sequences from analysis of human framework sequences, e.g., as described in Knappik et al, J.mol.biol.296:57-86, 2000).

The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions to promote stability or manufacturing).

As used herein, the term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This is achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart. See, e.g., morrison et al, proc.Natl.Acad.Sci.USA, 81; morrison and Oi, adv. Immunol.,44 (1988); verhoeyen et al, science,239 1534-1536 (1988); padlan, molec. Immun.,28, 489-498 (1991); padlan, molec. Immun.,31 (3): 169-217 (1994).

The term "recognize" as used herein refers to an antibody or antigen-binding fragment thereof that discovers and interacts with (e.g., binds) an epitope, whether the epitope is linear or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen-binding fragment of the invention specifically binds. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturants, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturants. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an Epitope include techniques in the art such as x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., epipope Mapping Protocols in Methods in Molecular Biology, vol 66, g.e.morris, editions (1996)).

The term "affinity" as used herein refers to the strength of the interaction of an antibody with an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen through weak non-covalent forces at multiple sites; the stronger the interaction, the stronger the affinity.

The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. However, an isolated antibody that specifically binds to one antigen may be cross-reactive with other antigens. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refers to those nucleic acids which encode identical or substantially identical amino acid sequences, or substantially identical sequences when the nucleic acid does not encode an amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. At each position where alanine is determined by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence herein encoding a polypeptide also describes every possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each such sequence.

With respect to polypeptide sequences, "conservatively modified variants" includes each substitution, deletion or addition to a polypeptide sequence which results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following group 8 contains amino acids that are conservative substitutions for each other: 1) Alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., creighton, proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

The term "optimized" as used herein means that the nucleotide sequence has been altered to encode an amino acid sequence using codons preferred in a production cell or organism, typically a eukaryotic cell, such as a yeast cell, a Pichia (Pichia) cell, a fungal cell, a Trichoderma (Trichoderma) cell, a chinese hamster ovary Cell (CHO), or a human cell. The optimized nucleotide sequence is engineered to retain, completely or to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence (which is also referred to as the "parent" sequence).

The term "percent identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over the full-length sequence, over a specified region or when not specified) when compared and aligned for maximum correspondence over a comparison window or within a specified region, as determined using the following sequence comparison algorithm or by manual alignment and visual inspection. Optionally, identity exists over a region of at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region of at least 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes reference to a segment of any number of contiguous positions selected from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. The optimal alignment of sequences for comparison is carried out, for example, by the local homology algorithm of Smith and Waterman, adv.Appl.Math.2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J.Mol.biol.48:443 (1970), by the similarity method of searching Pearson and Lipman, proc.Natl.Acad.Sci.USA 85 (1988), by the computerized implementation of these algorithms (Wisconsin Genetics Software Package, genetics Computer Group,575 Science Dr., madison, GAP, BESTFIT, FASTA and TFASTA in WI), or by manual alignment and visual inspection (see, for example, brent et al, current Protocols in Molecular Biology, 2003).

Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al, nuc.acids Res.25:3389-3402,1977; and Altschul et al, J.mol.biol.215:403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores were calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. When: (ii) the cumulative alignment score falls off its highest achieved value by an amount of X; (ii) the cumulative score reaches zero or less due to accumulation of one or more negative scoring residue alignments; or the end of any one sequence, the extension of the word hits in each direction is stopped. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses default word size (W) 11, expectation (E) 10, M =5, N = -4, and comparison of the two strands. For amino acid sequences, the BLASTP program aligns (B) 50, expect (E) 10, M =5, N = -4, and comparison of the two strands using default word size 3, and expect (E) 10 and BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) proc.natl.acad.sci.usa 89 10915).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., karlin and Altschul, proc. Natl. Acad. Sci. Usa 90. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller, comut.appl.biosci.4: 11-17,1988, which has been integrated into the ALIGN algorithm (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J.mol.biol.48:444-453, 1970) algorithms of the GAP program that have been integrated into the GCG software package (available on www.gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, and the

GAP weights

16, 14, 12, 10, 8, 6 or 4 and the

length weights

1, 2, 3, 4, 5 or 6.

In addition to the percentage of sequence identity indicated above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide Nucleic Acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses silent variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be accomplished by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues, as described in detail below (Batzer et al, (1991) Nucleic Acid Res.19:5081, ohtsuka et al, (1985) J.biol.chem.260:2605-2608; and Rossolini et al, (1994) mol.cell.Probes 8.

The term "operably linked" in the context of nucleic acids refers to a functional relationship of two or more polynucleotide (e.g., DNA) segments. Generally, it refers to the functional relationship between transcriptional regulatory sequences and transcribed sequences. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to transcribed sequences are physically adjacent to transcribed sequences, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent or in close proximity to the coding sequence (an enhancer enhances its transcription).

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to typical amino acid polymers as well as atypical amino acid polymers. Unless otherwise indicated, conservatively modified variants thereof are also encompassed by a particular polypeptide sequence.

The term "immunoconjugate" or "antibody conjugate" as used herein refers to the linkage of an antibody or antibody fragment thereof to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, or the like. The linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (many of which are known in the art) can be used to form immunoconjugates. In addition, the immunoconjugate may be provided in the form of a fusion protein, which may be expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein produced by joining two or more genes or gene fragments that originally encode separate proteins (including peptides and polypeptides). Fusion proteins are produced by ligation at the N or C terminus, or by insertion of a gene or gene fragment into a permissive region of one of the chaperone proteins. Translation of the fusion gene results in a single protein with functional properties from each of the original proteins.

The term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, except where indicated.

The term "cytotoxin" or "cytotoxic agent" as used herein refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy cells or malignancies.

The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

The terms "drug moiety" or "payload" are used interchangeably and refer to a chemical moiety conjugated to an antibody or antibody fragment of the invention, and may include any moiety used to attach an antibody or antibody fragment. For example, the drug moiety or payload can be an anti-cancer agent, an anti-inflammatory agent, an anti-fungal agent, an anti-bacterial agent, an anti-parasitic agent, an anti-viral agent, an anesthetic agent. In certain embodiments, the drug moiety is selected from the group consisting of a V-atpase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of the protein CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, an inhibitor of protein synthesis, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalating agent, a DNA minor groove binder, and a DHFR inhibitor. Suitable examples include auristatins, such as MMAE and MMAF; spinosyns, such as gamma-spinosyns; and maytansinoids, such as DM1 and DM4. Methods for attaching each of these to a linker compatible with the antibodies and methods of the invention are known in the art. See, e.g., singh et al, (2009) Therapeutic Antibodies: methods and Protocols, vol. 525, 445-457. Further, the payload can be a biophysical probe, fluorophore, spin label, infrared probe, affinity probe, chelator, spectroscopic probe, radioactive probe, lipid molecule, polyethylene glycol, polymer, spin label, DNA, RNA, protein, peptide, surface, antibody fragment, nanoparticle, quantum dot, liposome, PLGA particle, sugar or polysaccharide, reactive functional group, or a binder, surface, etc. that can link the conjugate to another moiety.

The term "drug-antibody ratio" (also referred to as "DAR") refers to the number of payloads or drug moieties of the antibodies that are linked to the immunoconjugate. For example, a ratio of drug antibodies of 2 represents the average of the two drug moieties bound to each antibody in the immunoconjugate sample. Some individual immunoconjugates with a drug-to-antibody ratio of 2 in the sample may have no or only one drug moiety attached; other immunoconjugates will have two, three, four or even more moieties on the individual antibodies in the sample. But the average value in the sample will be 2. There are different methods in the art for measuring the drug-antibody ratio of immunoconjugates.

In embodiments of the invention, the DAR in an immunoconjugate sample may be "homogeneous". A "homogeneous conjugate sample" is a sample with a narrow distribution of DAR. As an exemplary embodiment, in a homogeneous conjugated sample with a DAR of 2, unconjugated antibodies may be contained in the sample, and some antibodies with more than two DAR-conjugated moieties of about 2. By "a majority of the sample" is meant that at least more than 70%, or at least more than 80%, or at least more than 90% of the antibodies in the sample will be conjugated to both moieties.

As an exemplary embodiment, in a homogeneous conjugated sample with a DAR of 4, antibodies with more or less than four DAR-conjugated moieties of about 4 may be contained in the sample. By "a majority of the sample" is meant that at least more than 70%, or at least more than 80%, or at least more than 90% of the antibodies in the sample will be conjugated to the four moieties.

As an exemplary embodiment, in a homogeneous conjugated sample with a DAR of 6, antibodies with more or less than six DAR-conjugated moieties of about 6 may be contained in the sample. By "majority of the sample" is meant that at least more than 70%, or at least more than 80%, or at least more than 90% of the antibodies in the sample will be conjugated to the six moieties.

As an exemplary embodiment, in a homogeneous conjugated sample with a DAR of 8, some antibodies with less or more than eight DAR-conjugated moieties of about 4 may be contained in the sample. By "majority of the sample" is meant that at least more than 70%, or at least more than 80%, or at least more than 90% of the antibodies in the sample will be conjugated to the eight moieties.

An immunoconjugate having a "drug-to-antibody ratio of about 2" refers to a sample of such immunoconjugates in which the drug-to-antibody ratio can vary from about 1.6-2.4 moieties/antibody, 1.8-2.3 moieties/antibody, or 1.9-2.1 moieties/antibody.

An immunoconjugate having a "drug-to-antibody ratio of about 4" refers to a sample of such immunoconjugates in which the drug-to-antibody ratio can vary from about 3.6-4.4 parts per antibody, 3.8-4.3 parts per antibody, or 3.9-4.1 parts per antibody.

An immunoconjugate having a "drug-to-antibody ratio of about 6" refers to a sample of immunoconjugate in which the drug-to-antibody ratio may vary from about 5.6-6.4 parts per antibody, 5.8-6.3 parts per antibody, or 5.9-6.1 parts per antibody.

An immunoconjugate having a "drug-to-antibody ratio of about 8" refers to a sample of immunoconjugate in which the drug-to-antibody ratio may vary from about 7.6-84 parts per antibody, 7.8-8.3 parts per antibody, or 7.9-8.1 parts per antibody.

"tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

The term "anti-tumor activity" means a decrease in the proliferation rate, viability or metastatic activity of tumor cells. A possible way to show antitumor activity is to show a decrease or reduction in the growth rate of abnormal cells or tumor size stability that occurs during the course of treatment. This activity can be assessed using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to study anti-tumor activity.

The term "malignant tumor" refers to a non-benign tumor or cancer. As used herein, the term "cancer" includes malignancies characterized by unregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.

The term "cancer" includes primary malignancies (e.g., those tumors whose cells have not migrated to a site in the body of the subject other than the site of the primary tumor) and secondary malignancies (e.g., those tumors originating from metastases where tumor cells migrate to a second site different from the primary tumor).

As used herein, the term "optical isomer" or "stereoisomer" refers to any of a variety of stereoisomeric configurations, which may exist for a given compound of the present invention and includes geometric isomers. It is understood that the substituents may be attached at a chiral center at a carbon atom. The term "chiral" refers to a molecule that has non-superimposable properties on its mirror partner, while the term "achiral" refers to a molecule that is superimposable on its mirror partner. Thus, the present invention includes enantiomers, diastereomers, or racemates of the compounds. "enantiomers" are a pair of stereoisomers that are non-overlapping mirror images of each other. 1 of one pair of enantiomers: 1 the mixture is a "racemic" mixture. Where appropriate, the term is used to designate racemic mixtures. "diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry was assigned according to the Cahn-lngold-Prelog R-S system. When the compound is a pure enantiomer, the stereochemistry at each chiral carbon may be specified by R or S. Analytical compounds whose absolute configuration is unknown are designated (+) or (-), depending on the direction (dextro-or levorotatory) in which they rotate plane-polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which are defined in absolute stereochemistry as (R) -or (S) -.

Depending on the choice of the starting materials and processes, the compounds may be present in the form of one of the possible isomers or mixtures thereof, for example as pure optical isomers, or as isomer mixtures, such as racemic compounds and diastereoisomeric mixtures, depending on the number of asymmetric carbon atoms. The present invention is intended to include all such possible isomers, including racemic mixtures, diastereomeric mixtures and optically pure forms. Optically active (R) -and (S) -isomers can be prepared using chiral synthons or chiral reagents, or can be resolved using conventional techniques. If the compound contains a double bond, the substituent may be in the E or Z configuration. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may have either the cis or trans configuration. All tautomeric forms are also intended to be included.

As used herein, the term "salt" refers to an acid addition salt or a base addition salt of a compound of the present invention. "salt" includes in particular "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the present invention and which are generally not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be formed using inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, theophylline (chlorothioglycinate), citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecanoate, oleate, palmitate, embonate, phosphate/hydrophosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I through XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from the base moiety or the acid moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., na, ca, mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are generally carried out in water or an organic solvent, or a mixture of both. Generally, it may be desirable to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile where applicable. For example, the compounds can be found in "Remington's Pharmaceutical Sciences", 20 th edition, mack Publishing Company, easton, pa., (1985); and Stahl and Wermuth, of the "Handbook of Pharmaceutical Salts: properties, selection, and Use" (Wiley-VCH, weinheim, germany, 2002).

Any formula given herein is also intended to represent unlabeled as well as isotopically labeled forms of the compounds. Isotopically-labeled compounds have the structure depicted in the formulae given herein, except that one or more atoms are replaced by an atom of selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, respectively, such as ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ F、 ³¹ P、 ³² P、 ³⁵ S、 ³⁶ Cl、 ¹²⁵ I. The invention includes a plurality of isotopically-labelled compounds as defined herein, for example radioisotopes, such as ³ H and ¹⁴ those compounds in which C is present, or non-radioactive isotopes, e.g. ² H and ¹³ c are those compounds where C is present. Such isotopically labeled compounds are useful in metabolic studies (using ¹⁴ C) Reaction kinetics study (using, for example ² H or ³ H) Detection or imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays, or in the radiation treatment of patients. In particular, the method of manufacturing a semiconductor device, ¹⁸ f or labeled compounds are particularly desirable for PET or SPECT studies. Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and preparations using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously used.

In addition, the use of heavier isotopes, particularly deuterium (i.e., ² h or D) is takenGenerations may provide certain therapeutic advantages resulting from higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improvement in therapeutic index. It is understood that deuterium is considered in this context to be a substituent of the compound of formula (I). The concentration of the heavier isotopes, in particular deuterium, can be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the abundance of an isotope and the natural abundance of the specified isotope. If a substituent in a compound of the invention is indicated as deuterium, then the compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation on each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition Mack Printing Company,1990, pages 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, use of any conventional carrier in therapeutic or pharmaceutical compositions is contemplated.

The term "therapeutically effective amount" of a compound of the invention refers to an amount of a compound of the invention that will elicit the biological or medical response of a subject, e.g., a decrease or inhibition of enzyme or protein activity, or ameliorate symptoms, alleviate a condition, slow or delay the progression of a disease, or prevent a disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that is effective, when administered to a subject, to at least partially alleviate, inhibit, prevent and/or ameliorate a condition or disorder or disease, or to at least partially inhibit the activity of a target enzyme or receptor.

As used herein, the terms "inhibit", "inhibition" or "inhibiting" refer to the reduction or inhibition of a given condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

As used herein, the terms "treat", "treating" or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., slowing or preventing or reducing the occurrence of the disease or at least one clinical symptom thereof). In another embodiment, "treating," "treatment," or "treatment" refers to ameliorating or improving at least one physical parameter, including those that a patient may not be able to discern. In yet another embodiment, "treating," "treatment," or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or by both. In yet another embodiment, "treating," "treatment," or "treatment" refers to preventing or delaying the onset or occurrence or progression of a disease or disorder.

As used herein, a subject is "in need of" treatment if the subject would benefit biologically, medically or in quality of life from the treatment.

As used herein, the terms "a," "an," "the," and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term "mercapto-maleimide" as used herein describes a group formed by the reaction of a mercapto group with a maleimide, which has the general formula

Wherein Y and Z are groups to be linked by a thiol-maleimide linkage and may be a linker unit, and may be attached to an antibody or payload. In some cases, Y is an engineered antibody of the invention, and the sulfur atom shown in the formula is from a cysteine at one of the substitution sites described herein; and Z represents a joint unit connecting the payloads.

As used herein, "Linker Unit (LU)" refers to a covalent chemical linkage between two moieties, such as an antibody and a payload. Each LU may contain one or more components described herein, such as L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ . The linker unit may be selected to provide suitable spacing between the joined parts, or to provide certain physico-chemical properties, or to allow cleavage of the linker unit under certain conditions.

As used herein, "cleavable" refers to a linker or Linker Unit (LU) that connects two moieties by covalent attachment, but breaks down under physiological conditions to separate the covalent attachment between the moieties. Cleavage can be enzymatic or non-enzymatic, but the payload is typically released from the antibody without degrading the antibody.

As used herein, "non-cleavable" refers to a linker or Linker Unit (LU) that is not susceptible to cleavage under physiological conditions. While the linker may be physiologically modified, it remains payload-linked to the antibody until the antibody is substantially degraded, i.e., antibody degradation in vivo precedes linker cleavage.

As used herein, "cyclooctyne" refers to an 8-membered ring containing a carbon-carbon triple bond (acetylene). The ring optionally being fused to one or two benzene rings, which may be substituted by 1-4C _1-4 Alkyl radical, C _1-4 Alkoxy, halogen, hydroxy, COOH, COOL ₁ 、-C(O)NH-L ₁ 、O-L ₁ Or the like, and may contain N, O or S as a ring member. In a preferred embodiment, the cyclooctyne may be C ₈ Hydrocarbon rings, especially saturated split rings except triple bonds, and which may be substituted by F or hydroxyl, and may be connected to the linker or LU by-O-, -C (O), C (O) NH or C (O) O.

As used herein, "cyclooctyne" refers to an 8-membered ring containing at least one double bond, particularly a trans double bond.The ring optionally being fused to one or two benzene rings, which may be substituted by 1-4C _1-4 Alkyl radical, C _1-4 Alkoxy, halogen, hydroxy, COOH, COOL ₁ 、-C(O)NH-L ₁ 、O-L ₁ Or the like, and may contain N, O or S as a ring member. In a preferred embodiment, the cyclooctene can be an isolated C saturated except for the trans double bond ₈ A hydrocarbon ring, and may be optionally substituted with F or hydroxyl, and the linker or LU may be connected by-O-, -C (O), C (O) NH or C (O) O.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

Brief Description of Drawings

FIG. 1 is a graph of surface accessibility of amino acid residues in human IgG1 heavy (A) and kappa light (B) chains. Surface accessibility was calculated using Surface Racer 5.0 and expressed as square angstroms

FIG. 2 location of selected 92TAG mutation in the structure of human IgG1 with kappa light chain. The residues selected for TAG mutation are shown in black on only one of the two heavy chains and the selected residue is shown for one of the two kappa light chains (1hzh. An open source Molecular modeling software package PyMOL (The PyMOL Molecular Graphics System, version 1.5.0) was used.

LLC) display structure.

Figure 3 amino acid sequence alignment of the heavy chain constant region of trastuzumab and antibody 14090. Residues mutated to Cys in trastuzumab antibody and antibody 14090 are underlined. Amino acid residues in the heavy chain are numbered by the Eu numbering system (Edelman et al, 1969).

FIG. 4 alignment of amino acid sequences of the constant regions of trastuzumab, human IgG1, igG2, igG3, and IgG 4.

FIG. 5 amino acid sequence alignment of the constant regions of human kappa and lambda light chains. A. Residues mutated to Cys in the kappa light chain of trastuzumab and the lambda light chain of antibody 14090 are underlined. B. Selected residues for Cys mutation are shown in the PyMOL structural model of human lambda light chain (Protein Structure database entry 3G6D.pdb)

Figure 6 trastuzumab Cys antibody was analyzed by non-reducing SDS-PAGE.

FIG. 7 size exclusion chromatography of trastuzumab LC-S156C mutant antibody (dotted line) and wild-type trastuzumab (solid line).

FIG. 8 analysis of wild-type trastuzumab (A) and trastuzumab LC-E158C mutant antibody (B) by reverse phase high pressure liquid chromatography (RP-HPLC).

FIG. 9 MS analysis of trastuzumab LC-R108C mutant antibody after protein A purification (intact MS).

FIG. 10 shows the structure of MC-MMAF.

FIG. 11. Trastuzumab Cys antibody conjugation mix with MC-MMAF was analyzed by RP-HPLC. The RP-HPLC trace of the conjugate mixture is shown as a dashed line. The RP-HPLC traces of the unmodified antibody are shown as solid lines. LC-R108C-MMAF, B.HC-360C-MMAF, C.LC-S156C-MMAF, and D.HC-S275C-MMAF ADCs.

FIG. 12. Trastuzumab Cys antibody conjugation mix with MC-MMAF was analyzed by RP-HPLC. The RP-HPLC trace of the conjugate mixture is shown as a dashed line. The RP-HPLC trace of the unmodified antibody is shown as a solid line. HC-S134C-MMAF, and B.HC-S136C-MMAF ADCs.

FIG. 13 trastuzumab Cys-MMAF ADCs were analyzed by analytical size exclusion chromatography (AnSEC). Trastuzumab HC-K290C-MMAF ADC (short dashed line), trastuzumab LC-R142C-MMAF ADC (dashed line), and trastuzumab LC-L154C-MMAF ADC (dotted dashed line) were compared to unmodified wild-type trastuzumab (solid line).

FIG. 14 thermal melting profiles of unmodified wild-type trastuzumab and trastuzumab HC-T335C-MMAF, trastuzumab HC-S337C-MMAF and trastuzumab HC-K360C-MMAF ADCs.

Figure 15 cell proliferation assays for trastuzumab LC-S159C-MMAF and a.hcc1954, b.mda-MB231 clone 16 and c.mda-MB231 clone 40 cells.

FIG. 16 IC of trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 cell proliferation assay ₅₀ 。

Figure 17 cell proliferation assay for antibody 14090HC-S375C-MMAF ADC with a.cmk11-5 and b.jurkat cells.

Figure 18 pharmacokinetic studies of trastuzumab LC-Cys-MMAF ADCs that did not show significant drug loss. A. Wild-type unconjugated trastuzumab, b.lc-K107C-MMAF, c.lc-R108C-MMAF, d.lc-L154C-MMAF, and e.lc-S159C-MMAF ADC.

Figure 19. Pharmacokinetic studies of trastuzumab HC-Cys-MMAF ADCs that did not show significant drug loss. HC-K121C-MMAD, B.HC-L174C-MMAF, C.HC-E258C-MMAF, and D.HC-R292C-MMAF ADCs.

Figure 20. Pharmacokinetic study of trastuzumab Cys-MMAF ADCs demonstrating significant drug loss. LC-T129C-MMAF, B.LC-E143C-MMAF, C.HC-K246C-MMAF, and D.HC-R344C-MMAF ADCs.

Figure 21. Pharmacokinetic study of two trastuzumab Cys-MMAF ADCs demonstrating rapid clearance in vivo. HC-T335C-MMAF and B.HC-S337C-MMAF ADCs.

Figure 22 in vivo potency studies of trastuzumab Cys-MMAF ADCs in mda-MB231 clone 16 xenograft mouse model.

FIG. 23 residence time of trastuzumab Pcl MMAF DAR 2 ADCs as measured by hydrophobic interaction chromatography. ABA-MMAF is attached to the Pcl residues replacing the indicated HC or LC residues. A) HC-conjugated ADCs. B) LC conjugated ADCs. The retention time (WT) of unconjugated wild-type antibody is indicated.

FIG. 24 location of selected payload sites in the structure of human IgG1 with kappa light chain. Selected residues are shown in black on only one of the two heavy chains and selected residues for one of the two kappa light chains (1hzh. An open source Molecular modeling software package (The PyMOL Molecular Graphics System, version 1.5.0 was used.

LLC) display structureThree rotations.

Figure 25 pharmacokinetic studies of trastuzumab with

DAR

4, 6 and 8 and antibody 14090 Cys-MMAF ADCs prepared with antibodies with 2, 3 or 4 Cys mutations. DAR 4 trastuzumab ADCs HC-E258C-LC-S159C-MMAF (A), HC-S375C-LC-S159C-MMAF (B), HC-E258C-LC-E165C-MMAF (C), HC-S375C-LC-E165C-MMAF (D), HC-E152C-LC-R142C-MMAF (E), HC-P171C-LC-R142C-MMAF and HC-E152C-LC-S159C-MMAF (G); DAR 4 antibody 14090 ADCs: HC-S375C-LC-A143C-MMAF (H), HC-K360C-LC-V159C-MMAF (I), and HC-S375C-LC-V159C-MMAF (J); DAR 6 trastuzumab (ADCs) HC-K334C-S375C-LC-E165C-MMAF and HC-K334C-K392C-LC-E165C-MMAF; DAR 8 trastuzumab ADCs HC-K334C-K360C-S375C-LC-E165C-MMAF, HC-K334C-K360C-K392C-LC-E165C-MMAF and HC-K334C-S375C-K392C-LC-E165C-MMAF. Antibody 14090 is mouse cross-reactive and thus cleared more rapidly than trastuzumab ADC that does not bind any mouse antigen.

Detailed Description

The present invention provides methods for site-specifically labeling an antibody or antibody fragment by replacing one or more amino acids of a parent antibody or antibody fragment with a cysteine amino acid ("Cys") at a specific position, such that the engineered antibody or antibody fragment is capable of being conjugated to a variety of agents (e.g., cytotoxic agents). The invention also provides immunoconjugates produced by using the methods described herein.

When cysteine is engineered into a parent antibody or antibody fragment, the modified antibody or antibody fragment is first recovered from the expression medium, with the cysteine or Glutathione (GSH) attached to the engineered cysteine sites by disulfide bonds (Chen et al, (2009) mAbs 16, 353-571). The attached cysteine or GSH is then removed in a reduction step, which also reduces all the native interchain disulfide bonds of the parent antibody or antibody fragment. In the second step, these disulfide bonds are reoxidized before conjugation occurs. The present disclosure shows that when cysteine is engineered at certain sites, the re-oxidation step does not proceed smoothly, possibly due to incorrect disulfide bond formation. Thus, the present invention provides unique sets of sites on the antibody heavy chain constant region and antibody light chain constant region, respectively, where Cys substitution as described herein results in a modified antibody or antibody fragment that performs well in the reoxidation process, and also results in a stable and well-behaved immunoconjugate.

Site-specific antibody labeling of the invention can be accomplished using a variety of chemically readily available labeling agents, such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immunopotentiators, radiographic probes, prodrugs, and other molecules.

Accordingly, the present invention provides methods of preparing homogeneous immunoconjugates having defined drug-to-antibody ratios for use in cancer therapy and other indications as well as imaging agents. The invention also provides immunoconjugates prepared thereby, and pharmaceutical compositions comprising these immunoconjugates. The methods of the present invention can be used in combination with other conjugation methods known in the art.

The embodiments set forth below represent certain aspects and variations of the invention:

LU is a joint unit as described herein;

x is a payload or drug moiety;

and n is an integer from 1-16. In these embodiments, n is preferably about 2, about 4, about 6, or about 8.LU is usually a group of formula-L ₁ -L ₂ -L ₃ -L ₄ -L ₅ -L ₆ -, wherein L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Independently selected from A ₁ -、-A ₁ X ² -and-X ² -; wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、(O(C(R ⁴ ) ₂ ) _n ) _m -、

-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -, or (O (C (R) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、/>

-CHR ⁴ (CH ₂ ) _n C(＝O)NH-、-CHR ⁴ (CH ₂ ) _n NHC (= O) -, -C (= O) NH-, and NHC (= O) -;

each R ⁴ Independently selected from H, C _1-4 Alkyl, side chains of known amino acids, -C (= O) OH and-OH,

each R ⁵ Independently selected from H, C _1-4 C substituted by alkyl, phenyl or 1-3-OH groups _1-4 An alkyl group;

each R ⁶ Independently selected from H, fluorine, C (= O) OH substituted benzyloxy, -C (= O) OH substituted benzyl, C (= O) OH substituted C _1-4 Alkoxy and-C (= O) OH substituted C _1-4 An alkyl group;

R ⁷ independently selected from H, C _1-4 Alkyl, phenyl, pyrimidine and pyridine;

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

In some of these embodiments, the immunoconjugate comprises a set of formulae

Wherein the sulfur atom is the sulfur of a cysteine residue in the modified antibody or antibody fragment and is positioned at one of the substitution sites identified herein.

In any of the above embodiments, the cysteine substitution site may be a position corresponding to one of the positions identified by position numbering, even though the position of the position in the sequence has been altered by modification or truncation of the full-length antibody. The corresponding sites can be readily identified by aligning the antibody or fragment with a full-length antibody.

1. Site-specific cysteine engineered antibodies

Site-specific markers

Antibodies of the invention (e.g., parent antibodies, optionally containing one or more atypical amino acids) are numbered according to the EU numbering system set forth in Edelman et al, (1969) proc.natl.acad.usa 63, 78-85, except that the lambda light chain is numbered according to the Kabat numbering system set forth in Kabat et al, (1991) 5 th edition NIH Publication No. 91-3242. Human IgG1 constant regions are used as representative of the present application. However, the present invention is not limited to human IgG1; the corresponding amino acid positions can be easily deduced by sequence alignment. For example, fig. 4 shows an alignment of human IgG1, igG2, igG3, and IgG4 heavy chain constant regions such that Cys engineering sites identified in the IgG1 constant region can be readily identified for IgG2, igG3, and IgG4 as shown in fig. 4. For the light chain constant region, igG1, igG2, igG3, and IgG4 are identical. Table 1 below lists the amino acid positions in the constant region of the antibody heavy chain that may be replaced by cysteines. Table 2 lists the amino acid positions in the constant region of the kappa light chain of the antibody that may be replaced by cysteine. Table 3 lists the amino acid positions in the constant region of the lambda light chain of the antibody that can be replaced by cysteine.

Table 1 cysteine substitution sites identified in the heavy chain constant region of human IgG1 (sites numbered according to the EU numbering system).

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Table 2. Cysteine substitution sites identified in the kappa light chain constant region of human IgG1 (sites numbered according to the EU numbering system).

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Table 3. Cysteine substitution sites identified on the lambda light chain of human IgG 1.

The present discovery is not limited by any particular antibody or antibody fragment due to the high sequence homology of the constant regions of IgG1, igG2, igG3, and IgG4 antibodies.

In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises a substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in its heavy chain constant region selected from the positions identified in table 1. In a particular embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392 of the heavy chain. For example, the immunoconjugates of the invention comprise a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment thereof is at a position selected from the group consisting of positions 121 and 124, 121 and 152, 121 and 171, 121 and 174, 121 and 258, 121 and 292, 121 and 333, 121 and 334, 121 and 360, 121 and 375, 121 and 392, 124 and 152, 124 and 171, 124 and 174, 124 and 258, 124 and 292, 124 and 333, 124 and 334, 124 and 360, 124 and 375, 124 and 392,152 and 171, 152 and 174, 152 and 258, 152 and 292, 152 and 333, 152 and 334, of the heavy chain 152 and 360, 152 and 375, 152 and 392, 171 and 174, 171 and 258, 171 and 292, 171 and 333, 171 and 360, 171 and 375, 174 and 258, 174 and 292, 174 and 333, 174 and 334, 174 and 360, 174 and 375, 174 and 392, 258 and 292, 258 and 333, 258 and 334, 258 and 360, 258 and 375, 258 and 392, 292 and 333, 292 and 334, 292 and 360, 292 and 375, 292 and 392, 333 and 334, 333 and 360, 333 and 375, 333 and 392;334 and 360, 334 and 375, 334 and 392, 360 and 375, 360 and 392 or 375 and 392 comprise a substitution of two amino acids with cysteine in their constant regions.

In another embodiment, the immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment thereof is substituted at a position selected from positions 121, 124 and 152 of the heavy chain; 121. 124 and 171; 121. 124 and 174; 121. 124 and 258; 121. 124 and 292; 121. 124 and 333; 121. 124 and 334; 121. 124 and 360; 121. 124 and 375; 121. 124 and 392; 121. 152 and 171; 121. 152 and 174; 121. 152 and 258; 121. 152 and 292; 121. 152 and 333; 121. 152 and 334; 121. 152 and 360; 121. 152 and 375; 121. 152 and 392; 121. 171 and 174; 121. 171 and 258; 121. 171 and 292; 121. 171 and 333; 121. 171 and 334; 121. 171 and 360; 121. 171 and 375; 121. 171 and 392; 121. 174 and 258, 121, 174 and 292; 121. 174 and 333; 121. 174, and 334; 121. 174 and 360; 121. 174 and 375; 121. 174, and 392; 121. 258 and 292; 121. 258 and 333; 121. 258 and 334; 121. 258 and 360; 121. 258 and 375; 121. 258 and 392; 121. 292 and 333; 121. 292 and 334; 121. 292 and 360; 121. 292 and 375; 121. 292 and 392; 121. 333 and 334; 121. 333, and 360; 121. 333 and 375; 121. 333, and 392; 121. 334 and 360; 121. 334 and 375; 121. 334 and 392; 121. 360 and 375; 121. 360 and 392; 121. 375 and 392; 124. 152 and 171; 124. 152 and 174; 124. 152 and 258; 124. 152 and 292; 124. 152 and 333; 124. 152 and 334; 124. 152 and 360; 124. 152 and 375; 124. 152 and 392; 124. 171 and 174; 124. 171 and 258; 124. 171 and 292; 124. 171 and 333; 124. 171 and 334; 124. 171 and 360; 124. 171 and 375; 124. 171 and 392; 124. 174 and 258; 124. 174, and 292; 124. 174 and 333; 124. 174 and 334; 124. 174 and 360; 124. 174 and 375; 124. 174, and 392; 124. 258 and 292; 124. 258 and 333; 124. 258 and 334; 124. 258 and 360; 124. 258 and 375; 124. 258 and 392; 124. 292 and 333; 124. 292 and 334; 124. 292 and 360; 124. 292 and 375; 124. 292 and 392; 124. 333, and 360; 124. 333 and 334; 124. 333 and 375; 124. 333, and 392; 124. 334 and 360; 124. 334 and 375; 124. 334 and 392; 124. 360 and 375; 124. 360 and 392; 124. 375 and 392; 152. 171 and 174; 152. 171 and 258; 152. 171 and 292; 152. 171 and 333; 152. 171 and 334; 152. 171 and 360; 152. 171 and 375; 152. 171 and 392; 152. 174 and 258; 152. 174 and 292; 152. 174 and 333; 152. 174 and 334; 152. 174 and 360; 152. 174 and 375; 152. 174 and 392; 152. 258 and 292; 152. 258 and 333; 152. 258 and 334; 152. 258 and 360; 152. 258 and 375; 152. 258 and 392; 152. 292 and 333; 152. 292 and 334; 152. 292 and 360; 152. 292 and 375; 152. 292 and 392; 152. 333 and 334; 152. 333, and 360; 152. 333 and 375; 152. 333, and 392; 152. 334 and 360; 152. 334 and 375; 152. 334 and 392; 152. 360 and 375; 152. 360 and 392; 152. 375 and 392; 171. 174 and 258; 171. 174 and 292; 171. 174 and 333; 171. 174 and 334; 171. 174 and 360; 171. 174 and 375; 171. 174 and 392; 171. 258 and 292; 171. 258 and 292; 171. 258 and 333; 171. 258 and 334; 171. 258 and 360; 171. 258 and 375; 171. 258 and 392; 171. 292 and 333; 171. 292 and 334; 171. 292 and 360; 171. 292 and 375; 171. 292 and 392; 171. 333 and 334; 171. 333, and 360; 171. 333 and 375; 171. 333 and 392; 171. 334 and 360; 171. 334 and 392; 171. 360 and 375; 171. 360 and 392; 171. 375 and 392; 174. 258 and 292; 174. 258 and 333; 174. 258 and 334; 174. 258 and 360; 174. 258 and 375; 174. 258 and 392; 174. 292 and 333; 174. 292 and 334; 174. 292 and 360; 174. 292 and 375; 174. 292 and 392; 174. 333 and 334; 174. 333, and 360; 174. 333 and 375; 174. 333, and 392; 174. 334 and 360; 174. 334 and 375; 174. 334 and 392; 174. 360 and 375; 174. 360 and 392; 174. 375 and 392; 258. 292 and 333; 258. 292 and 334; 258. 292 and 360; 258. 292 and 375; 258. 292 and 392; 258. 333, and 360; 258. 333 and 375; 258. 333, and 392; 258. 334 and 360; 258. 334 and 375; 258. 334 and 392; 258. 360 and 375; 258. 360 and 392; 258. 375 and 392; 292. 333 and 334; 292. 333, and 360; 292. 333 and 375; 292. 333 and 392; 292. 334 and 360; 292. 334 and 375; 292. 334 and 392; 292. 360 and 375; 292. 360 and 392; 292. 375 and 392; 333. 334 and 360; 333. 334 and 375; 333. 334 and 392; 333. 360 and 375, 333, 360 and 392; 333. 375 and 392; 334. 360 and 375; 334. 360 and 392; or 360, 375, and 392 comprising a substitution of three amino acids with cysteine on their constant regions.

In one embodiment, the immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment is attached to the heavy chain at a position selected from the group consisting of positions 152, 333, 375, and 392 of the heavy chain; or 152, 334, 375 and 392 comprising a cysteine to four amino acid substitution on its constant region.

In particular embodiments, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises

SEQ ID NO

2, 3, 9, 11, 12, 13, 14, 16, 21, 25, 26, 28, 30, 31, 32, 33, 34, 36, 38, 39, 40, 43, 44, 45, 46, 47, 51, 53, 54, 56, 57, or 60. In another specific embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises

SEQ ID NO

6, 7, 8, 15, 19, 20, 22, 23, 24, 27, 36, 37, 41, 49, 52, 55, 58, or 59.

In another embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in its light chain constant region at a position selected from those identified in table 2. In a particular embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at positions 107, 108, 142, 145, 159, 161, and 165 selected from a light chain, wherein said light chain is a human kappa light chain. For example, the immunoconjugates of the invention comprise an immunoconjugate of a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment is present at a position selected from positions 107 and 108 of the light chain; 107 and 142;107 and 145;107 and 159;107 and 161;107 and 165;108 and 142;108 and 145;108 and 159;108 and 161;108 and 165;142 and 145;142 and 159;142 and 161;142 and 165;145 and 159;145 and 161;145 and 165;159 and 161;159 and 165;161 and 165 comprises a cysteine-for-two amino acid substitution in its constant region, wherein the light chain is a human kappa light chain. In another embodiment, the immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment is attached to the light chain at a position selected from the group consisting of positions 107, 108 and 142; 107. 108 and 145; 107. 108 and 159; 107. 108 and 161; 107. 108 and 165; 107. 142 and 145; 107. 142 and 159; 107. 142 and 161; 107. 142 and 165; 107. 145 and 159; 107. 145 and 161; 107. 145 and 165; 107. 159 and 161; 107. 159 and 165; 107. 161 and 165; 108. 142 and 145; 108. 142 and 159; 108. 142 and 161; 108. 142 and 165; 108. 145 and 159; 108. 145 and 161; 108. 145 and 165; 108. 159 and 161; 108. 159 and 165; 108. 161 and 165; 142. 145 and 159; 142. 145 and 161; 142. 145 and 165; 142. 159 and 161; 142. 159 and 165; 142. 161 and 165; 145. 159 and 161; 145. 159 and 165; 145. 161 and 165; or 159, 161 and 165, wherein the light chain is a human kappa light chain.

In certain embodiments, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises SEQ ID NO 63, 65, 68, 70, 72, 73, 74, 78, 79, 80, 81, 82, 83, 86, 87, or 88. In another embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises SEQ ID NO 64, 66, 67, 84, 85, or 89 63, 64, 65, 66, 67, 68, 70, 72, 73, 74, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89.

In another embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its light chain constant region selected from the positions identified in table 3. In a particular embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 143, 147, 159, 163 and 168 of a light chain, wherein the light chain positions are numbered according to the Kabat system, and wherein the light chain is a human λ light chain. For example, an immunoconjugate of the invention comprises an immunoconjugate of a modified antibody or antibody fragment thereof and a drug moiety, wherein the modified antibody or antibody fragment is at a position selected from positions 143 and 147 of a light chain; 143 and 159;143 and 163;143 and 168;147 and 159;147 and 163;147 and 168;159 and 163;159 and 168; or 163 and 168 comprising a substitution of two amino acids with cysteine on its constant region, wherein the light chain positions are numbered according to the Kabat system, and wherein the light chain is a human lambda light chain. In another embodiment, the immunoconjugate of the invention comprises an immunoconjugate of a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment is attached to the light chain at a position selected from positions 143, 147 and 159; 143. 147 and 163; 143. 147 and 168; 143. 159 and 163; 143. 159 and 168; 143. 163 and 168; 147. 159 and 163; 147. 159 and 168; 147. 163 and 168; or 159, 163 and 168 comprising a substitution of three amino acids with cysteine on its constant region, wherein the light chain positions are numbered according to the Kabat system, and wherein the light chain is a human lambda light chain.

In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein the modified antibody or antibody fragment thereof comprises SEQ ID NOs 92, 94, 96, 97, or 98. In another specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO 93 or 95.

In one embodiment, the immunoconjugate may have a DAR of about 2 or about 4. In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein the modified antibody or antibody fragment comprises a Cys substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in its heavy chain constant region selected from the positions identified in table 1, and a Cys substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in its light chain constant region selected from the positions identified in table 2 or table 3. In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment comprises a Cys substitution of one or more amino acids in its heavy chain constant region selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392; and a Cys substitution comprising one or more amino acids in its light chain constant region selected from positions 107, 108, 142, 145, 159, 161, and 165, wherein the light chain is a human kappa light chain. In one embodiment, the modified antibody or antibody fragment of the invention may comprise a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 107 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 108 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 142 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 145 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 159 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 161 of the human kappa light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain. In one embodiment, the modified antibody or antibody fragment of the invention comprises Cys substitutions at positions 375 and 392 of a heavy chain, and Cys substitutions at position 165 of a human kappa light chain. In one embodiment, the modified antibody or antibody fragment of the invention may comprise Cys substitutions at positions 334 and 375 of the heavy chain, and Cys substitutions at position 165 of the human kappa light chain. In another embodiment, the modified antibody or antibody fragment of the invention may comprise a Cys substitution at position 334 and position 392 of the heavy chain, and a Cys substitution at position 165 of the human kappa light chain. In one embodiment, those combined immunoconjugates can have a DAR of about 4 or about 6.

In one embodiment, the modified antibody or antibody fragment of the invention may comprise Cys substitutions at positions 334, 375, and 392 of a heavy chain, and Cys substitutions at position 165 of a human kappa light chain. In one embodiment, the modified antibody or antibody fragment of the invention may comprise Cys substitutions at positions 333, 375, and 392 of a heavy chain, and Cys substitutions at position 165 of a human kappa light chain. In one embodiment, those combinations may have a DAR of about 4, 6, or 8.

In one embodiment, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment comprises a Cys substitution of one or more amino acids in its heavy chain constant region selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392; and a Cys substitution comprising one or more amino acids in its light chain constant region selected from positions 143, 147, 159, 163, and 168, wherein the light chain is a human λ light chain. For example, a modified antibody or antibody fragment of the invention may comprise a Cys substitution at position 121 of a heavy chain, and a Cys substitution at position 143 of a human λ light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 121 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 124 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 152 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 171 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 174 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 258 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 292 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 333 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 334 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 360 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 375 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 143 of the human λ light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 147 of the human λ light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 159 of the human λ light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 163 of the human λ light chain; or a Cys substitution at position 392 of the heavy chain, and a Cys substitution at position 168 of the human λ light chain.

In an embodiment of the invention, the amino acid substitution described herein is a cysteine comprising a thiol group. In some aspects of the invention, thiol groups are utilized for chemical conjugation and attachment to Linker Units (LU) and/or drug moieties. In some embodiments, the immunoconjugate of the invention comprises a drug moiety selected from the group consisting of a V-atpase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, dolastatin, a maytansinoid, metAP (methionine aminopeptidase), an inhibitor of nuclear export of the protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalating agent, a DNA minor groove binding agent, and a DHFR inhibitor. In some embodiments, the immunoconjugates of the invention comprise a drug moiety that is an anti-cancer agent. The modified antibodies or antibody fragments of the invention can be in any form known in the art, such as monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibodies or antibody fragments thereof.

According to the invention, the modified antibody heavy and/or light chain (or antibody fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8 or more cysteine substitutions in its constant region. In one embodiment, the modified antibody or antibody fragment contains 2, 4, 6, 8 or more cysteine substitutions in its constant region. In some embodiments, the modified antibody, antibody fragment thereof, or immunoconjugate comprises 2 or 4 Cys substitutions.

In one embodiment, the parent antibody (antibody without cysteine substitutions) is an IgG, igM, igE or IgA antibody. In a particular embodiment, the parent antibody is an IgG1 antibody. In another specific embodiment, the parent antibody is an IgG2, igG3 or IgG4 antibody.

The invention also provides a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids in its heavy chain constant region selected from the positions identified in table 1. In some embodiments, the present invention provides a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids in its light chain constant region selected from the positions identified in table 2 or table 3.

In certain embodiments, the modified antibodies or antibody fragments provided herein are labeled using the methods of the present invention in combination with other conjugation methods known in the art, including, but not limited to, chemoselective conjugation via lysine, histidine, tyrosine, formyl-lysine, pyrrolysine, pyrroline-carboxy-lysine, unnatural amino acids, and protein tags (e.g., S6 tags) for enzyme-mediated conjugation.

2. Conjugation chemistry

The conjugated antibodies or antibody fragments thereof provided herein are generated by post-translational modification of at least one cysteine residue incorporated into the above-described antibodies or antibody fragments thereof by a site-specific labeling method. Conjugated antibodies or antibody fragments can be prepared by methods known in the art for conjugating a payload of interest to a naturally occurring cysteine residue in a protein, and by the methods described for conjugating to a protein engineered to contain an additional cysteine residue in place of another amino acid of the native protein sequence.

In certain embodiments, the modified antibodies or antibody fragments thereof provided herein are conjugated using known methods, wherein the incorporated cysteine (Cys) is conjugated to a maleimide derivative, as in scheme Ia below. The modified antibodies of the invention that undergo this type of conjugation contain a thiol-maleimide linkage.

Conjugation via thiol-maleimide adduct formation

Wherein:

LU is a joint unit (LU), and

x is a payload or drug moiety.

In other embodiments, incorporated into a polyesterCys in the modified antibody or antibody fragment may be conjugated by reaction with an α -halocarbonyl compound, such as chloro, bromo, or iodo acetamide as shown in scheme Ib below. It will be appreciated that other leaving groups than halogen, such as tosylate, triflate and other alkyl or arylsulfonates may be used as the leaving group Y. Although scheme Ib describes the reaction of Cys thiol with α -haloacetamide, the method includes alkylation of the incorporated Cys sulfur with a group of the formula Y-CHR-C (= O) -, where R is H or C _1-4 Alkyl, Y is a leaving group (typically C1, br or I, and optionally an alkyl sulfonate or aryl sulfonate); it is not limited to amides.

Conjugation by reaction with α -halocarbonyl compounds.

Y is a leaving group (Cl, br, I, otf, etc.)

LU is a joint unit

X is a payload or drug moiety

Alternatively, cys incorporated into the modified antibody or antibody fragment may be conjugated by reaction with an external thiol group under conditions that induce the formation of a disulfide bond between the external thiol group and the sulfur atom of the incorporated cysteine residue shown in scheme Ic below. In these examples, R may be H; however, it has been found that compounds in which one or both of the R groups represent an alkyl group, such as methyl, increase the stability of the disulfide.

Conjugation via disulfide formation scheme ic.

Each R is independently H or C _1-4 Alkyl radical

LU is a joint unit

X is a payload or drug moiety

Such post-translational modifications are set forth above in schemes (Ia) - (Ic) by way of example only, where the starting structure represents a cysteine incorporated into the light or heavy chain of an antibody at one of the specific sites identified herein. Methods for performing each of these conjugation methods are well known in the art. Antibodies can be modified in their light chain, or their heavy chain, or both light and heavy chains by these methods. An antibody in which each light chain or each heavy chain has been modified to contain a single incorporated cysteine will generally contain two conjugation sites, since an antibody typically contains two light chains and two heavy chains.

When conjugated, the modified antibodies of the invention typically contain 1-12, often 2-8, and preferably 2, 4, or 6-LU-X (linker unit-payload) moieties. In some embodiments, the antibody light or heavy chain is modified to incorporate two new Cys residues in two of the specific sites identified herein for the Cys substitutions (or, one Cys is incorporated into the light chain and one is incorporated into the heavy chain), so the tetrameric antibody ultimately contains four conjugation sites. Likewise, an antibody may be modified by replacing 3 or 4 of its natural amino acids with Cys at a particular site identified herein in the light or heavy chain or combinations thereof, resulting in 6 or 8 conjugation sites in a tetrameric antibody.

X in these conjugates represents a payload, which can be any chemical moiety used to attach antibodies. In some embodiments, X is a drug moiety selected from cytotoxins, anti-cancer agents, anti-inflammatory agents, antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents, immunopotentiators, and anesthetic agents or any other therapeutic agent, or a biologically active moiety or drug moiety. In another embodiment, X is a label, such as a biophysical probe, fluorophore, affinity probe, spectroscopic probe, radioactive probe, spin label, or quantum dot. In other embodiments, X is a chemical moiety that modifies the physicochemical properties of the antibody, such as a lipid molecule, polyethylene glycol, polymer, polysaccharide, liposome, or chelator. In other embodiments, X is a functional or detectable biomolecule, such as a nucleic acid, ribonucleic acid, protein, peptide (e.g., an enzyme or receptor), sugar or polysaccharide, an antibody, or an antibody fragment. In other embodiments, X is an anchoring moiety, such as a nanoparticle, PLGA particle, or surface, or any binding moiety for specifically binding the conjugate to another moiety, such as a histidine tag, poly G, biotin, avidin, streptavidin, and the like. In other embodiments, X is a reactive functional group that can be used to attach the antibody conjugate to another chemical moiety, such as a drug moiety, a label, another antibody, another chemical moiety, or a surface.

The Linker Unit (LU) can be any suitable chemical moiety that covalently attaches a thiol-reactive group (e.g., maleimide, α -halocarbonyl, vinylcarbonyl (e.g., acrylate or acrylamide), vinylsulfone, vinylpyridine, or thiol) to the payload. Many suitable LU's are known in the art. For example, LU may comprise what is referred to herein as L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ One, two, three, four, five, six or more linkers. In certain embodiments, the LU comprises a linker selected from a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, a photo-cleavable linker, or any combination thereof, and optionally contains a self-degradation (self-immolative) spacer.

In some embodiments, LU is of formula-L ₁ -L ₂ -L ₃ -L ₄ -or-L ₁ -L-L ₃ -L ₄ -L ₅ -L ₆ -a group of (a). Linking group L for LU ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Including alkylene- (CH) ₂ ) _n - (where n is 1-20, typically 1-10 or 1-6), ethylene glycol units (-CH) ₂ CH ₂ O-) _n (wherein n is 1-20, typically 1-10 or 1-6), amide-C (= O) -NH-or-NH-C (= O) -, ester-C (= O) -O-or-O-C (= O) -, a ring with two available attachment points such as divalent phenyl, C _3-8 Cycloalkyl or C _4-8 Heterocyclyl, amino acid-NH-CHR-C = O-or-C (= O) -CHR-NH-, wherein R is a side chain of a known amino acid (often one of the typical amino acids, but also including examples Such as norvaline, norleucine, homoserine, homocysteine, phenylglycine, citrulline, and other named alpha amino acids), polypeptides of known amino acids (e.g., dipeptides, tripeptides, tetrapeptides, etc.), thiol-maleimide linkages (from the addition of SH to maleimide), -S-CR2-, and other thiol ethers such as-S-CR 2-C (= O) -or-C (= O) -CR2-S-, where R is as defined above for scheme Ic, -CH2-C (= O) -, and disulfides (-S-), as well as combinations of any of these with other linkers described below, such as bonds, non-enzymatically cleavable linkers, non-cleavable linkers, enzymatically cleavable linkers, photostable linkers, photo-cleavable linkers, or linkers comprising self-degrading spacers.

In some embodiments, when LU is-L ₁ -L-L ₃ -L ₄ -L ₅ -L ₆ When is, L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ May be selected from:

-A ₁ -,-A ₁ X ² -and-X ² -; wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -,-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or- (O (C (R) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、

-CHR ⁴ (CH ₂ ) _n C(＝O)NH-,-CHR ⁴ (CH ₂ ) _n NHC (= O) -, -C (= O) NH-, and NHC (= O) -;

each R ⁶ Independently selected from H, fluorine, benzyloxy substituted by C (= O) OH, benzyl substituted by C (= O) OH, C (= O) OH _1-4 Alkoxy and-C (= O) OH substituted C _1-4 An alkyl group;

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

In some embodiments, L is ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Is a stable or non-cleavable linker. In some embodiments, L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Is cuttableA head, which can be cleaved chemically (hydrazone, disulfide) or enzymatically. In some embodiments, the enzymatically cleavable linker is a linker that is readily cleaved by a peptidase: the dipeptide Val-Cit linker of the two known amino acids (valine-citrulline) is one such linker. In another embodiment, the enzymatically cleavable linker is one triggered by the activity of glucuronidase:

are examples of such linkers, which also comprise a self-degrading spacer that spontaneously separates under physiological conditions upon glycosidic bond cleavage by a glucuronidase.

In some embodiments, the immunoconjugates of the invention comprise a modified cysteine residue of formula IIA or IIB:

wherein-CH ₂ -S-represents the side chain of a Cys incorporated at one of the selected Cys substitution sites described herein, and L ₂ –L ₆ And X represents a linking group and a payload, respectively, as further described herein. In some embodiments of IIA, L is a bond. In some embodiments of IIB, L ₂ Is NH or O. In some embodiments of IIA and IIB, L ₃ Is selected from (CH) ₂ ) _1-10 And (CH) ₂ CH ₂ O) _1-6 。L ₄ 、L ₅ And L ₆ Are additional optional linkers selected from those described herein. In certain embodiments, L ₆ May be a carbonyl group (C = O) or a linker comprising a self-degrading spacer.

In certain embodiments, the Linker Unit (LU) is-L ₁ –L ₂ –L ₃ –L ₄ -, wherein:

L ₁ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linkerA linker, photostable linker or photocleavable linker of (a);

L ₂ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, or a photocleavable linker;

L ₃ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker or a photo-cleavable linker, and

L ₄ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, a photocleavable linker or a linker comprising a self-degrading spacer.

In certain embodiments, the Linker Unit (LU) is-L ₁ –L ₂ –L ₃ –L ₄ -, wherein

L ₁ A non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, or a photocleavable linker;

In some embodiments of the LU, L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Is a cleavable linker, and the LU is considered to be cleavable. Also, in some embodiments of LU, L ₁ 、L ₂ 、L ₃ 、L ₄ 、L ₅ And L ₆ Is a non-cleavable linker. In certain of these embodiments, each linker of the LU isNon-cleavable, and the LU is considered non-cleavable.

In some of the above embodiments, wherein LU is-L ₁ –L ₂ –L ₃ –L ₄ –，L ₁ 、L ₂ 、L ₃ And L ₄ At least one of which is selected from-A ₁ -、-A ₁ X ² -and-X ² -a linker of (a); wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or- (O (C (R)) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、

CHR ⁴ (CH ₂ ) _n C(＝O)NH-、-CHR ⁴ (CH ₂ ) _n NHC (= O) -, -C (= O) NH-, and NHC (= O) -;

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

In these embodiments, the other linkers of the LU are independently selected from the group consisting of a bond, A ¹ -、-A ₁ X ² -、-X ² Non-enzymatically cleavable linkers, non-cleavable linkers, enzymatically cleavable linkers, photostable linkers, photo-cleavable linkers and linkers comprising self-degrading spacers.

L ₁ Is a bond, -A ₁ -、-A ₁ X ² -or-X ² -; wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or- (O (C (R) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、

each R ⁶ Independently selected from H, fluorine, -C (= O) OH substituted benzyloxy, -C (= O) OH substituted benzyl, -C (= O) OH substituted C _1-4 Alkoxy and-C (= O) OH substituted C _1-4 An alkyl group;

R ⁸ is selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

L ₄ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, a photocleavable linker, or a linker comprising a self-degrading spacer.

In certain embodiments, L ₁ Is C (= O) -CH ₂ CH ₂ -NH-C(＝O)-CH ₂ CH ₂ -S-, so LU is-C (= O) -CH ₂ CH ₂ -NH-C(＝O)-CH ₂ CH ₂ -S-L ₂ -L ₃ -L ₄ -。

L ₁ Is a bond, -A ₁ -、-A ₁ X ² -or-X ² -; wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、/>

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

L ₃ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker, or a photocleavable linker;

In certain embodiments, the Linker Unit (LU) is-L ₁ –L ₂ –L ₃ –L ₄ -, in which

L ₁ Is a bond, -A ₁ -、-A ₁ X ² -or-X ² -; wherein:

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、/>

each R ⁶ Independently selected from H, fluoro, -C (= O) OH substituted benzyloxy, -C (= O) OH substituted benzyl, -C (= O) OH substituted C _1-4 Alkoxy and-C (= O) OH substituted C _1-4 An alkyl group;

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

L ₃ Is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photostable linker or a photocleavable linker, and

L ₁ Is a bond, -A ₁ -、-A ₁ X ² -or-X ² -; wherein:

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、/>

R ⁸ is selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

L ₂ is a bond, a non-enzymatically cleavable linker or a non-cleavable linker;

L ₃ is a bond, a non-enzymatically cleavable linker or a non-cleavable linker;

L ₄ is a bond, an enzymatically cleavable linker or a linker comprising a self-degrading spacer.

L ₁ Is a bond, -A ₁ -、-A ₁ X ² -or-X ² -；

L ₂ Is a bond, -A ₂ -or-A ₂ X ² -；

L ₃ Is a bond, -A ₃ -or-A ₃ X ² -；

L ₄ Is a bond, -A ₄ -、-A ₄ X ² -、

/>

A ₁ are-C (= O) NH-, -NHC (= O) -, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or- (O (C (R) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

A ₂ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NR ⁴ -、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n S-、-(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n -、-S(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH-、-(C(R ⁴ ) ₂ ) _n NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m OC(＝O)NH(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m OC(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、

A ₃ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n S-、-(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n -、-S(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m OC(＝O)NH(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m OC(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m OC(＝O)-、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m OC(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m C(＝O)-、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m C(＝O)-、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、

A ₄ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-(O(C(R ⁴ ) ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((C(R ⁴ ) ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(((C(R ⁴ ) ₂ ) _n O) _m C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-(CH ₂ ) _n NHC(＝O)-、-(C(R ⁴ ) ₂ ) _n NHC(＝O)-、-NHC(＝O)(CH ₂ ) _n -、-NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-C(＝O)NH(C(R ⁴ ) ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-S(C(R ⁴ ) ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)NH(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-C(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(C(R ⁴ ) ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n (O(C(R ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NHC(＝O)(C(R ⁴ ) ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(C(R ⁴ ) ₂ ) _n NH((C(R ⁴ ) ₂ ) _n O) _m (C(R ⁴ ) ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or- (O (C (R) ⁴ ) ₂ ) _n ) _m NHC(＝O)(C(R ⁴ ) ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、

R ⁸ is selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

L ₁ Is a bond, -A1-, -A ₁ X ² -or-X ² -；

L ₂ Is a bond, -A ₂ -or-A ₂ X ² -；

L ₃ Is a bond, -A ₃ -or-A ₃ X ² -；

L ₄ Is a bond, -A ₄ -、-A ₄ X ² -、

/>

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

A ₂ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or

A ₃ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or

A ₄ -C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n -、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)NH-、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n S-、-S(CH ₂ ) _n C(＝O)NH-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-C(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n C(＝O)-、-(CH ₂ ) _n (O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond,

-S-、-Si(OH) ₂ O-、

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

L ₁ Is a bond, -A1-, -A ₁ X ² -or-X ² -；

L ₂ Is a bond, -A ₂ -or-A ₂ X ² -；

L ₃ Is a bond, -A ₃ -or-A ₃ X ² -；

L ₄ Is a bond, -A ₄ -、-A ₄ X ² -、

/>

A ₁ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

A ₂ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or

A ₃ is-C (＝O)NH-、-C(＝O)NH(CH ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -、-(O(CH ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -or

A ₄ are-C (= O) NH-, -C (= O) NH (CH) ₂ ) _n -、-C(＝O)NH(CH ₂ ) _n S-、-(O(CH ₂ ) _n ) _m -、-((CH ₂ ) _n O) _m (CH ₂ ) _n -、-NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NHC(＝O)-、-C(＝O)NH(CH ₂ ) _n NHC(＝O)(CH ₂ ) _n -、-(CH ₂ ) _n NH((CH ₂ ) _n O) _m (CH ₂ ) _n -or- (O (CH) ₂ ) _n ) _m NHC(＝O)(CH ₂ ) _n -；

Each X ² Independently selected from the group consisting of a bond, R ⁸ 、

/>

-S-、-Si(OH) ₂ O-、/>

R ⁸ is independently selected from

R ⁹ Independently selected from H and C _1-6 A haloalkyl group;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

In one embodiment, L ₁ Is- (CH) ₂ ) _1-10 -C (= O) -, e.g. - (CH) ₂ ) ₅ -C (= O) -; and L is ₂ 、L ₃ And L ₄ Each representing a bond.

In certain embodiments, LU comprises a val-cit linker of the formula, wherein X represents a payload, typically a drug moiety, such as one having anti-cancer activity:

when L is ₄ -L ₅ -L ₆ When it is a val-cit linker as shown above, L ₃ Is preferably- (CH) ₂ ) _2-6 -C(＝O)-。

In certain embodiments, the X group is a maytansinoid, such as DM1 or DM4, or a dolastatin analog or derivative, such as dolastatin 10 or 15 and auristatin MMAF or MMAE, or a calicheamicin such as N-acetyl- γ -calicheamicin, or a label or dye, such as rhodamine or tetramethyl rhodamine.

As used herein, a "linker" is any chemical moiety capable of linking an antibody or fragment thereof to an X group (payload) to form an immunoconjugate. The linker may be susceptible to cleavage, such as acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the compound or antibody retains activity. Alternatively, the joint may be substantially resistant to cutting. The linker may or may not include a self-degrading spacer.

As used herein, X ¹ Non-limiting examples of non-enzymatically cleavable linkers in which groups are conjugated to the modified antibodies or antibody fragments thereof provided herein include acid labile linkers, linkers containing disulfide moieties, linkers containing triazole moieties, linkers containing hydrazone moieties, linkers containing thioether moieties, linkers containing diazo moieties, linkers containing oxime moieties, linkers containing amide moieties, and linkers containing acetamide moieties.

Non-limiting examples of enzymatically cleavable linkers that conjugate an X group to a modified antibody or antibody fragment thereof provided herein, as used herein, include, but are not limited to, linkers cleaved by a protease, linkers cleaved by an amidase, and linkers cleaved by a β -glucuronidase or another glycosidase.

In certain embodiments, such enzyme-cleavable linkers are linkers cleaved by cathepsins, including cathepsin Z, cathepsin B, cathepsin H, and cathepsin C. In certain embodiments, the enzymatically cleavable linker is a dipeptide cleaved by a cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin B, cathepsin H, or cathepsin C. In certain embodiments, the enzymatically cleavable linker is a cathepsin B cleavable peptide linker. In certain embodiments, the enzymatically cleavable linker is a cathepsin B cleavable dipeptide linker. In certain embodiments, the enzymatically cleavable dipeptide linker is valine-citrulline or phenylalanine-lysine. Other non-limiting examples of enzymatically cleavable linkers that conjugate an X group to a modified antibody or antibody fragment thereof provided herein, as used herein, include, but are not limited to

The linker cleaved by the beta-glucuronidase, for example,

see Ducry et al, bioconjugate Chem, (2010) vol.21 (1), 5-13.

A "self-degrading spacer" is a bifunctional chemical moiety covalently linked at one end to a first chemical moiety and at the other end to a second chemical moiety, thereby forming a stable three-part molecule. The linker may comprise a self-degrading spacer to which a third chemical moiety is bonded, which third chemical moiety may be chemically or enzymatically cleaved from the spacer. After the bond between the self-degrading spacer and the first chemical moiety or the third chemical moiety is cleaved, the self-degrading spacer undergoes a rapid and spontaneous intramolecular reaction and is thereby separated from the second chemical moiety. These intramolecular reactions typically involve electronic rearrangement, such as 1,4 or 1,6 or 1,8 elimination or cyclization to form highly advantageous five-or six-membered rings. In certain embodiments of the invention, the first or third moiety is an enzyme cleavable group and the cleavage results from an enzymatic reaction, while in other embodiments, the first or third moiety is an acid labile group and the cleavage occurs as a result of a change in pH. As applied to the present invention, the second moiety is a "payload" group as defined herein. In certain embodiments, cleavage of the first or third moiety from the self-degrading spacer results from cleavage by a proteolytic enzyme, while in other embodiments it results from cleavage by a hydrolytic enzyme. In certain embodiments, cleavage of the first or third moiety from the self-degrading spacer results from cleavage by a cathepsin or glucuronidase.

In certain embodiments, the enzyme cleavable linker is a peptide linker and the self-degrading spacer is covalently linked to the peptide linker at one end thereof and to the drug moiety at the other end thereof. The three-part molecule is stable in the absence of the enzyme and pharmacologically inactive, but can be enzymatically cleaved at the bond covalently linking the spacer moiety and the peptide moiety. The peptide moiety is cleaved from the three-part molecule, which initiates the self-degradation feature of the spacer moiety, resulting in spontaneous cleavage of the bond covalently linking the spacer moiety and the drug moiety, thereby effecting release of the pharmacologically active form of the drug.

In other embodiments, the linker comprises a self-degrading spacer that is directly or indirectly attached to the peptide at one end and a payload at the other end; and the spacer is attached to a third moiety, which can be enzymatically cleaved from the spacer, for example, by a glucuronidase. Upon cleavage of the third portion, the spacer degrades or rearranges in a manner that causes the payload to be released. An example of a linker with this type of self-degrading spacer is such a glucuronidase-cleavable linker, in which hydrolysis of the acetal catalyzed by glucuronidase releases a phenolic compound that spontaneously decomposes under physiological conditions:

Optionally for the addition of X ¹ Non-limiting examples of moieties conjugated to self-degrading spacers of modified antibodies or antibody fragments thereof provided herein include, but are not limited to including benzylA carbonyl moiety, a ditolyl ether moiety, a 4-aminobutyrate moiety, a hemidisclosure moiety, or a moiety of an N-acylhemidisclosure moiety.

Other examples of self-degrading spacers include, but are not limited to, p-aminobenzyloxycarbonyl, aromatic compounds with electron similarity to p-aminobenzyloxycarbonyl, such as 2-aminoimidazole-5-methanol derivatives and o-or p-aminobenzyl acetals. In certain embodiments, self-degrading spacers, which undergo cyclization after hydrolysis of the amide bond, as used herein include substituted and unsubstituted 4-aminobutanoic acid amides and 2-aminophenylpropionic acid amides.

In certain embodiments, the self-degrading spacer is

In yet other embodiments, the self-degrading spacer is

Wherein n is 1 or 2. In other embodiments, the self-degradation spacer is ∑ er>

Wherein n is 1 or 2. In other embodiments, the self-degradation spacer is ÷ or ÷ in>

Wherein n is 1 or 2. In other embodiments, the self-degrading spacer is

Wherein n is 1 or 2.

Schemes (2 a-2 c) illustrate the modified antibodies or antibody sheets thereof provided hereinPost-translational modification of a segment, wherein Linker Unit (LU) is-L ₁ –L ₂ –L ₃ –L ₄ And L1 is in each case a group which reacts with the neo-Cys.

Scheme 2a.

Scheme 2b.

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Scheme 3c.

In each of schemes 2a-2c, the starting material is a replacement Cys residue in an antibody or antibody fragment modified as described herein, wherein the dashed bond indicates attachment to a contiguous residue of the antibody or antibody fragment; each R is H or C _1-4 Alkyl, typically H or methyl; l is ₂ 、L ₃ And L ₄ Are components of the connection unit LU, such as those described above; x is the payload; and is connected to L ₂ The radical of sulfur with the substituent Cys according to the invention is L ₁ 。

In some embodiments of the invention, X is a reactive functional group that can be linked to another chemical moiety by an antibody conjugated through interaction with a suitable complementary functional group. Table 4 describes some examples of reactive functional groups that X may represent with complementary functional groups that may be used to link a conjugate comprising X with another compound. Methods for using X to attach corresponding complementary functional groups are well known in the art. Ligation using azide compounds is typically accomplished using either 'Click' or copper-free Click chemistry; reactions involving hydrazines, alkoxyamines or acyl hydrazines are generally carried out by forming a schiff base with one of the carbonyl functions.

TABLE 4

Exemplary ligation products prepared using these components are described in Table 5, wherein Y ¹ Represents an antibody of the invention, A ₁ Representing the linking antibody to payload X ^a A Linking Unit (LU) of formula II-a, -L ₂ -L ₃ -L ₄ -represents a compound which can be present in the molecule via X ^a Linker unit to which conjugated antibody is attached, and X ¹ Representing the payload. Payload X ^a Is a reactive functional group, and X on formula II-a ^b Are the corresponding complementary functional groups, and formula II-a itself represents the molecule to which the conjugated antibody is to be attached. The third column in Table 5 depicts the data from X ^a And X ^b The product of the reaction.

TABLE 5

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In certain embodiments, the modified antibodies or antibody fragments thereof provided herein are conjugated at an "X group-to-antibody" (payload-to-antibody) ratio of about 1-16, such as 1-12, or 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibodies or antibody fragments thereof contain 1, 2, 3, 4, 5, 6, 7, or 8 cysteine residues incorporated at the specific sites disclosed herein. For example, an "X group-to-antibody" ratio of 4 can be achieved by incorporating two Cys residues into the heavy chain of the antibody, which will contain 4 conjugation sites, two from each heavy chain. Immunoconjugates of such antibodies will contain up to 4 payload groups, which may be the same or different and preferably all the same. In another example, an "X group-to-antibody" ratio of 4 can be achieved by incorporating one Cys residue into the heavy chain of the antibody and a second Cys residue into the light chain, resulting in 4 conjugation sites, two in the two heavy chains and two in the two light chains. Ratios of 6, 8, or higher can be achieved by a combination of 3, 4, or more cysteine substitutions in the heavy and light chains of the antibody. Substitution of multiple cysteine groups into the antibody can lead to improper disulfide formation and other problems. Thus to load more than 4 payload groups onto one antibody molecule, the methods of the invention may alternatively be conjugated with methods that do not rely on reactions on cysteine sulfur, such as acylation on lysine, or by S6-tag or pci methods.

Although the payload to antibody ratio has an accurate value for a particular conjugated molecule, it will be appreciated that when used to describe a sample containing many molecules, typically in the conjugation step, the value is often an average value due to some degree of heterogeneity. The average loading of the immunoconjugate samples is referred to herein as the drug-to-antibody ratio, or DAR. In some embodiments, the DAR is between about 1 and about 16, typically about 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least 50% of the samples by weight are compounds having an average ratio of plus or minus 2, and preferably, at least 50% of the samples are conjugates containing an average ratio of plus or minus 1. Preferred embodiments include immunoconjugates wherein the DAR is about 2 or about 8, e.g., about 2, about 4, about 6 or about 8. In some embodiments, a DAR of 'about n' means that the measurement for the DAR is within 10% of n (in formula (I)).

Other alterations of the framework of the Fc region

The present invention provides site-specifically labeled immunoconjugates. The immunoconjugates of the invention can comprise a modified antibody or antibody fragment thereof, further comprising a p-V _H And/or V _L Modification of internal framework residues, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues into the corresponding germline sequence. More particularly, an antibody that has been subjected to somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. In order to restore the framework sequences to their germline configuration, somatic mutations can be "back-mutated" into germline sequences, for example by site-directed mutagenesis. The invention is also intended to include such "back-mutated" antibodies.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in further detail in U.S. patent publication No. 20030153043 to Carr et al.

In addition to or as an alternative to modifications made within the framework or CDR regions, the antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter glycosylation thereof, again altering one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. The process is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. For example, the number of cysteine residues in the hinge region of CH1 is altered to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More particularly, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired staphylococcal (staphylocccyl) protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In yet another embodiment, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be replaced with a different amino acid residue, such that the antibody has an altered affinity for an effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand for which the affinity is altered may be, for example, an Fc receptor or the C1 component of complement. Such methods are described, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

In another embodiment, one or more amino acids selected from the group consisting of amino acid residues may be replaced by a different amino acid residue, such that the antibody has altered C1q binding and/or reduced or abolished Complement Dependent Cytotoxicity (CDC). This method is described, for example, in U.S. Pat. No. 6,194,551 to Idusogene et al.

In another embodiment, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This method is described, for example, in PCT publication WO 94/29351 to Bodmer et al. In particular embodiments, one or more amino acids of an antibody or antibody fragment thereof of the invention are replaced with one or more heterotypic amino acid residues, such as those shown in fig. 4 for the IgG1 subclass and the kappa isotype. Heterotypic amino acid residues also include, but are not limited to, the constant regions of the heavy chains of the IgG1, igG2, and IgG3 subclasses, and the constant regions of the light chains of the kappa isotype, as described by Jefferis et al, MAbs.1:332-338 (2009).

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the fey receptor by modifying one or more amino acids. This method is described, for example, in PCT publication WO 00/42072 to Presta. In addition, the binding sites for human IgG1 to Fc γ Rl, fc γ RII, fc γ RIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al, J.biol.chem.276:6591-6604, 2001).

In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. For example, glycosylation can be altered to increase the affinity of an antibody for an "antigen". Such carbohydrate modifications may be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made which result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at such sites. Such aglycosylation may increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies can be made with altered types of glycosylation, such as low fucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of the antibody. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 to Hang et al describes cell lines having a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in the cell line exhibit low fucosylation. PCT publication WO 03/035835 to Presta describes a variant CHO cell line, lecl3 cell, which has a reduced ability to attach fucose to Asn (297) linked carbohydrates, and also results in low fucosylation of antibodies expressed in the host cell (see also Shields et al, (2002) J.biol. Chem.277: 26733-26740). PCT publication WO 99/54342 to Umana et al describes cell lines engineered to express a glycoprotein-modified glycosyltransferase (e.g., β (1, 4) -N acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures that result in increased ADCC activity of the antibody (see also Umana et al, nat. Biotech.17:176-180, 1999).

In another embodiment, the antibody is modified to increase its biological half-life. A variety of approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, or T256F, as described in U.S. patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, it may be in CH1 or C _L Intraregionally altered antibodies to contain salvage receptor binding epitopes obtained from two loops of the CH2 domain of the Fc region of IgG, as described in Presta et al, U.S. patent nos. 5,869,046 and 6,121,022.

4. Antibody conjugates

The present invention provides site-specific labeling methods, modified antibodies and antibody fragments thereof, and immunoconjugates made thereby. Using the methods of the invention, the modified antibody or antibody fragment thereof can be conjugated to a label, such as a drug moiety, e.g., an anti-cancer agent, an autoimmune therapeutic agent, an anti-inflammatory agent, an anti-fungal agent, an anti-bacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent, or an imaging agent, such as a chelator for PET imaging, or a fluorescent label, or an MRI contrast agent. It is also possible to use several identical or different labeling moieties in combination with the method of the invention for conjugating the antibody or antibody fragment with other conjugation methods.

In certain embodiments, the immunoconjugate of the invention comprises a drug moiety selected from the group consisting of a V-atpase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, dolastatin, a maytansinoid, metAP (methionine aminopeptidase), an inhibitor of nuclear export of the protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalating agent, a DNA minor groove binder, a topoisomerase inhibitor, an RNA synthesis inhibitor, a kinesin inhibitor, an inhibitor of protein-protein interactions, and a DHFR inhibitor.

In addition, the modified antibodies or antibody fragments of the invention may be conjugated to a drug moiety that modifies a given biological response. The drug moiety is not to be understood as being limited to classical chemotherapeutic agents. For example, the drug moiety may be an immune modulator, such as an immunopotentiator, a small molecule immunopotentiator, a TLR agonist, a CpG oligomer, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope peptide, and the like. The drug moiety may also be an oligonucleotide, siRNA, shRNA, cDNA, or the like. Alternatively, the drug moiety may be a protein, peptide or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, apoptotic agents, anti-angiogenic agents, or biological response modifiers such as, for example, lymphokines.

In one embodiment, the modified antibody or antibody fragment of the invention is conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Examples of cytotoxins include, but are not limited to, taxanes (see, e.g., international (PCT) patent publication Nos. WO 01/38318 and PCT/US 03/02675), DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g., sasse et al, J.Antibiot. (Tokyo), 53,879-85 (2000), suzawa et al, bioorg.Med.Chem.,8,2175-84 (2000), ichimura et al, J.Antibiot. (Tokyo), 44,1045-53 (1991), francisco et al, blood (2003) (electronic publication prior to print publication), U.S. Pat. Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. patent application publication No. 2001/0036923A1, pending U.S. patent application Ser. Nos. 10/024,290 and 10/116,053, and International (PCT) patent application No. WO 01/49698), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, doxorubicin, daunorubicin, dihydroxyanthramycin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, and puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), ablative agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin (former daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (former actinomycin), bleomycin, mithramycin and Amphenomycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) (see, for example, seattle Genetics US 20090721).

Other examples of therapeutic cytotoxins that can be conjugated to the modified antibodies or antibody fragments of the present invention include duocarmycin, calicheamicin, maytansine, and auristatin, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg) ^Tm ；Wyeth-Ayerst)。

For further discussion of the type of cytotoxin, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito et al, (2003) adv. Drug delivery. Rev.55:199-215; trail et al, (2003) Cancer immunol.immunoher.52: 328-337; payne, (2003) Cancer Cell 3; allen, (2002) nat. Rev. Cancer 2; pasan and Kreitman, (2002) curr. Opin. Investig. Drugs 3; senter and Springer, (2001) adv. Drug Deliv. Rev.53:247-264.

According to the invention, the modified antibody or antibody fragment thereof may also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceuticalAn agent, referred to as a radioimmunoconjugate. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ^l31 Indium, indium ¹¹¹ Yttrium, yttrium ⁹⁰ And lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radioactive immunoconjugates are commercially available and include Zevalin ^TM (DEC Pharmaceuticals) and Bexxar ^TM (Corixa Pharmaceuticals) and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. In certain embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N, N ', N ", N'" -tetraacetic acid (DOTA), which can attach antibodies through a linker molecule. Such linker molecules are generally known in the art and are described in Denadro et al, (1998) Clin. Cancer Res.4 (10): 2483-90; peterson et al, (1999) bioconjugate. Chem.10 (4): 553-7; and Zimmerman et al, (1999) Nucl. Med. Biol.26 (8): 943-50, each incorporated herein by reference in its entirety.

The invention also provides modified antibodies or fragments thereof that specifically bind to an antigen. The modified antibody or fragment may be conjugated or fused to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce a fusion protein. In particular, the invention provides compositions comprising antibody fragments (e.g., fab fragments, fd fragments, fv fragments, F (ab) 2 fragments, V) as described herein _H Fragment, V _H CDR、V _L Domain or V _L CDR) and heterologous proteins, polypeptides or peptides.

In some embodiments, a modified antibody fragment without antigen binding specificity, such as but not limited to a modified Fc domain with an engineered cysteine residue of the invention, is used to produce a fusion protein comprising the antibody fragment (e.g., an engineered Fc) and a heterologous protein, polypeptide, or peptide.

Additional fusion proteins can be produced by techniques of gene shuttling, motif shuttling, exon shuttling, and/or codon shuttling (collectively "DNA shuttling"). DNA shuttling can be used to alter the activity of an antibody or fragment thereof of the invention (e.g., an antibody or fragment thereof having a higher affinity and a lower dissociation constant). See generally U.S. Pat. nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; patten et al, (1997) curr. Opinion Biotechnol.8:724-33; harayama, (1998) Trends Biotechnol.16 (2): 76-82; hansson et al (1999) J.mol.biol.287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24 (2): 308-313 (each of these patents and publications is incorporated by reference in its entirety). The antibody or fragment thereof, or the encoded antibody or fragment thereof, may be altered prior to recombination by random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies or fragments thereof that specifically bind to an antigen may be combined with one or more components, motifs, moieties (sections), moieties (parts), domains, fragments, etc. of one or more heterologous molecules.

In addition, the modified antibodies or antibody fragments thereof of the invention may be conjugated to marker sequences, such as peptides, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, inc.,9259 Eton Avenue, chatsworth, CA, 91311), many of which are commercially available. As described in Gentz et al, (1989) proc.natl.acad.sci.usa 86. Other peptide tags for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al, (1984) Cell 37. According to the invention, the antibody or antibody fragment may also be conjugated to a tumor penetrating peptide to enhance its efficacy.

In other embodiments, the modified antibodies or antibody fragments of the invention are conjugated to diagnostic or detectable agents. Such immunoconjugates can be used to monitor or prognose the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. The diagnosis can be accomplished by coupling the antibody with a detectable substance And detection, the detectable substance including but not limited to a variety of enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, alexa Fluor 350, alexa Fluor 405, alexa Fluor 430, alexa Fluor 488, alexa Fluor 500, alexa Fluor 514, alexa Fluor 532, alexa Fluor 546, alexa Fluor 555, alexa Fluor 568, alexa Fluor 594, alexa Fluor 610, alexa Fluor 633, alexa Fluor 647, alexa Fluor 660, alexa Fluor 680, alexa Fluor 700, alexa Fluor 750, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; the amount of radioactive material that is present, such as but not limited to iodine (I) ¹³¹ I、 ¹²⁵ I、 ¹²³ I. And ¹²¹ i), carbon (C) ¹⁴ C) Sulfur (S), (S) ³⁵ S), tritium ( ³ H) Indium (I) and (II) ¹¹⁵ In、 ¹¹³ In、 ¹¹² In, and ¹¹¹ in, technetium (C) ((C)) ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga、 ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F)、 ¹⁵³ Sm、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁹⁰ Y、47Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ⁶⁴ Cu、 ¹¹³ Sn, and ¹¹⁷ sn; and positron-generating metal and non-radioactive paramagnetic metal ions using a plurality of positron emission tomography.

The modified antibodies or antibody fragments of the invention may also be attached to a solid support, which is particularly useful for immunoassay or purification of a target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

5. Pharmaceutical composition

To prepare a pharmaceutical or sterile composition comprising the immunoconjugate, the immunoconjugate of the invention is admixed with a pharmaceutically acceptable carrier or excipient. The composition may additionally contain one or more other therapeutic agents suitable for treating or preventing cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, stomach cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors (e.g., schwannoma), head and neck cancer, bladder cancer, esophageal cancer, barrett's esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign Prostatic Hyperplasia (BPH), gynacomastica, and endometriosis).

Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers, for example in The form of lyophilized powders, slurries, aqueous solutions, emulsions or suspensions (see, for example, hardman et al, goodman and Gilman's The Pharmaceutical Basis of Therapeutics, mcGraw-Hill, new York, N.Y.,2001 Gennaro, remington.

The choice of administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In certain embodiments, the administration regimen maximizes the therapeutic dose delivered to the patient, consistent with an acceptable level of side effects. Thus, the amount of biological agent delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of Antibodies, cytokines and small molecules may be obtained (see, e.g., wawrynczak, antibody Therapy, bios Scientific pub. Ltd., oxfordshire, UK,1996, kresina (eds.), monoclonal Antibodies, cytokines and Arthrotis, marcel Dekker, new York, N.Y.,1991 Bach (eds.), monoclonal Antibodies and Peptide Therapy in Autoimmu Diseases, marcel Dekker, new York, N.Y.,1993, baert et al, new Engl. J. Med.348:601-608, mil2003, new Engl. J. 341: 1966-341, N.Y.,1993, new Engl. J. Med.348: 601-24, et al, ser. 76, ben. J. 35, EP J. 2000, EP J. Wo, EP 159J. 792.3, no. 24, EP 2000J. Wo, EP 15932.

Determination of the appropriate dosage is made by a clinician, for example, using parameters or factors known or suspected in the art to affect treatment or to be predictive of affecting treatment. Generally, the dosage is started in an amount somewhat less than the optimal dosage, which is then increased in small increments until the desired or optimal effect is achieved with respect to any adverse side effects. Important diagnostic measures include, for example, those of the symptoms of inflammation or the levels of inflammatory cytokines produced.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied to obtain an effective amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition employed, the age, sex, body weight, condition, general health and past medical history of the patient being treated, and like factors known in the medical arts.

Compositions comprising an antibody or fragment thereof of the invention may be provided by continuous infusion, or by intermittent doses, for example 1-7 times a day, week or week. The dosage may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally or by inhalation. A particular dosage regimen is one that involves a maximum dose or frequency of doses to avoid significant unwanted side effects.

For the immunoconjugates of the invention, the dose administered to the patient can be from 0.0001mg/kg to 100mg/kg of patient body weight. The dosage may be between 0.0001mg/kg to 20mg/kg, 0.0001mg/kg to 10mg/kg, 0.0001mg/kg to 5mg/kg, 0.0001mg/kg to 2mg/kg, 0.0001mg/kg to 1mg/kg, 0.0001mg/kg to 0.75mg/kg, 0.0001mg/kg to 0.5mg/kg, 0.0001mg/kg to 0.25mg/kg, 0.0001mg/kg to 0.15mg/kg, 0.0001mg/kg to 0.10mg/kg, 0.001 mg/kg to 0.5mg/kg, 0.01 mg/kg to 0.25mg/kg, or 0.01 mg/kg to 0.10mg/kg of patient body weight. The dose of the antibody or fragment thereof of the present invention can be calculated using the kilogram (kg) patient body weight multiplied by the dose to be administered in mg/kg.

The dosage of the immunoconjugate of the invention may be repeated and administration may be spaced at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart. In a specific embodiment, the dose of the immunoconjugate of the invention is repeated every 3 weeks.

An effective amount for a particular patient may vary depending on such factors as the condition being treated, the overall health of the patient, the method of administration and the severity of side effects (see, e.g., maynard et al, A Handbook of SOPs for Good Clinical Practice, interpharm Press, boca Raton, fla.,1996, dent, good Laboratory and Good Clinical Practice, urch publication, london, UK, 2001).

The route of administration may be by, for example, topical or epidermal application, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional or by injection or infusion by sustained release systems or implants (see, e.g., sidman et al, biopolymers 22, 547-556,1983, langer et al, j.biomed.mater.res.15:167-277,1981, lamer, chem.tech.12, 98-105,1982 epstein et al, proc.natl.acad.sci.usa 82, 3688-3692,1985 hwang et al, proc.natl.acad.sci.usa 77 4030-4034,1980; U.S. patent nos. 6,350,466 and 6,316,024). If desired, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the site of injection. In addition, pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and using formulations of aerosol. See, e.g., U.S. Pat. nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety.

The compositions of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. The skilled artisan will appreciate that the route and/or manner of administration will vary depending on the desired result. The routes of administration selected for the immunoconjugates of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, and is generally by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, the immunoconjugate of the invention is administered by infusion. In another embodiment, the immunoconjugate of the invention is administered subcutaneously.

If the immunoconjugates of the invention are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; sefton, CRC Crit. Ref biomed. Eng.14:20,1987, buchwald et al, surgery 88, 507, saudek et al, N.Engl. J.Med.321:574, 1989). Polymeric materials may be used to achieve Controlled or sustained Release of the treatment of the present invention (see, e.g., medical Applications of Controlled Release, langer and Wise (eds.), CRC Pres., boca Raton, fla., 1974. Examples of polymers for use in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyglycolide (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in the sustained release formulation are inert, free of leachable impurities, storage stable, sterile, and biodegradable. Controlled or sustained Release systems can be placed in close proximity to a prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., goodson, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138, 1984).

Controlled release systems are discussed in reviews by Langer, science 249 1527-1533, 1990. Any technique known to those skilled in the art can be used to produce sustained suitable formulations comprising one or more immunoconjugates of the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, ning et al, radiotherpy & Oncology 39, 179-189,1996; song et al, PDA Journal of Pharmaceutical Science & Technology 50, 372-397,1995; cleek et al, pro.int' l.Symp.control.Rel.Bioact.Mater.24:853-854,1997; and Lam et al, proc. Int' l. Symp. Control Rel. Bioact. Mater.24:759-760,1997, each of which is incorporated herein by reference in its entirety.

If the immunoconjugate of the invention is administered topically, they may be formulated as ointments, creams,Transdermal patches, emulsions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms known to those skilled in the art. See, e.g., remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19 th edition, mack pub. Co., easton, pa. (1995). For non-spray topical dosage forms, viscous to semi-solid or solid forms containing a carrier or one or more excipients compatible with topical application and having a kinetic viscosity (in some cases higher than water) are typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are sterile or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for affecting a variety of properties, such as, for example, osmotic pressure, if desired. Other suitable topical dosage forms include sprayable aerosol formulations wherein the active ingredient is in some cases packaged in combination with a solid or liquid inert carrier in combination with a pressurized volatile (e.g., a propellant gas, such as Freon) ^TM ) In a mixture or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms, if desired. Examples of such additional ingredients are well known in the art.

If the immunoconjugate-containing composition is administered intranasally, it may be formulated in the form of an aerosol, spray, liquid spray, or drops. In particular, prophylactic or therapeutic agents for use in accordance with the present invention can be conveniently delivered in a form delivered from pressurized packs or an aerosol spray from a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (made, for example, of gelatin) containing a powder mix of the compound and a suitable powder base, such as lactose or starch, may be formulated for use in an inhaler or insufflator.

Methods of co-administration with or treatment with a second therapeutic agent, such as cytokines, steroids, chemotherapeutic agents, antibiotics or radiation are known in The art (see, e.g., hardman et al, (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10.sup. Th., mcGraw-Hill, new York, N.Y.; poole and Peterson (eds.) (2001) Pharmacological assays for Advanced Therapeutics: A Practical Approach, lippincott, williams & Wilkins, phila., pa.; chamner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapeutics, lippincott, williams & Wilkins, phila., pa.; chabner and Longo (eds.) (2001). An effective amount of the therapeutic agent can reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40% or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with an immunoconjugate of the invention can be administered less than 5 minutes, less than 30 minutes, 1 hour, about 1 to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, about 12 hours to 18 hours, 18 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 hours to 52 hours, 52 hours to 60 hours, 60 hours to 72 hours, 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours to 120 hours apart from an immunoconjugate of the invention. Two or more therapies may be administered in the same patient visit.

In certain embodiments, the immunoconjugates of the invention can be formulated to ensure proper distribution in vivo. For example, the Blood Brain Barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (when required), they may be formulated, for example, in liposomes. For methods of making liposomes, see, e.g., U.S. Pat. nos. 4,522,811;5,374,548; and 5,399,331. Liposomes can comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, e.g., ranade, (1989) j. Clin. Pharmacol.29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannoside (Umezawa et al, (1988) biochem. Biophys. Res. Commun.153: 1038); antibodies (Bloeman et al (1995) FEBS Lett.357:140; the surfactant protein A receptor (Briscoe et al (1995) am.J.Physiol.1233: 134); p 120 (Schreier et al, (1994) J.biol.chem.269: 9090); see also k.keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; killion; i.j. fidler (1994) immunoassays 4.

The present invention provides regimens for administering to a subject in need thereof a pharmaceutical composition comprising an immunoconjugate of the invention alone or in combination with other therapies. The treatment (e.g., prophylactic or therapeutic agent) of the combination therapy of the invention can be administered to the subject simultaneously or sequentially. Treatment (e.g., prophylactic or therapeutic agents) of the combination therapies of the invention can also be administered cyclically. Cycling therapy involves administering a first treatment (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by a second treatment (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating the sequential administration, i.e., cycling, to reduce the development of resistance to one treatment (e.g., agent), to avoid or reduce the side effects of one treatment (e.g., agent), and/or to improve the efficacy of the treatment.

A treatment (e.g., prophylactic or therapeutic agent) of a combination therapy of the invention can be administered to a subject concurrently.

The term "simultaneously" is not limited to administration of treatments (e.g., prophylactic or therapeutic agents) at exactly the same time, but it also means that a pharmaceutical composition comprising an antibody or fragment thereof of the invention is administered to a subject sequentially and at time intervals, such that the antibody of the invention can act together with the other treatment or treatments to provide an increased benefit over if they were otherwise administered. For example, each treatment may be administered to the subject simultaneously or sequentially in any order at different time points; however, if not administered simultaneously, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each treatment may be administered to the subject separately in any suitable form and by any suitable route. In various embodiments, the subject is administered a treatment (e.g., a prophylactic or therapeutic agent) less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, 24 hours, 48 hours, 72 hours, or 1 week apart. In other embodiments, two or more treatments (e.g., prophylactic or therapeutic agents) are administered in the same patient visit.

The prophylactic or therapeutic agents of the combination therapy may be administered to the subject in the same pharmaceutical composition. Alternatively, prophylactic or therapeutic agents of a combination therapy may be administered to a subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agents can be administered to the subject by the same or different routes of administration.

Having fully described the invention, it is further illustrated by the following examples and claims, which are illustrative and not intended to be further limiting.

Examples

Example 1 selection of surface accessible sites for Cys mutation in human IgG1 heavy and kappa light chains

Surface exposed residues in the constant regions of human IgG1 heavy and human kappa light chains were identified in hIgG 1/kappa antibodies (Protein database structure 1HZH. Pdb, table 6, table 7, FIG. 1) using the computer program Surface 5.0 described by Tscobikov et al, "A novel computer program for Surface interaction calculation of access and molecular Surface areas and Surface course current," J.Compout.chem., 23,600-609 (2002). 88 residues were selected for Cys substitution, 59 positions in the hIgG heavy chain and 29 positions in the human kappa light chain based on the following criteria: 1) Selecting residues in the CH1, CH2 and CH3 domains of the heavy and light chain constant regions; 2) Selecting surface exposed residues but avoiding all exposed residues and C-terminal regions to avoid formation of dimers between antibodies; 3) Focusing on polar or charged residues, such as Ser, thr, lys, arg, glu, and Asp; and 4) excluding residues in the FcRn binding domain, the protein A binding domain, and the heavy chain hinge region.

Criteria 1), i.e. the selection of Cys substitution sites in the constant region of the antibody, ensures transferability of the conjugation site to a number of different antibodies. Criteria 2) observation of inter-antibody dimer formation based on the residues predominantly exposed for Cys substitution (residues excluded based on this criteria are listed in table 6). Based on the IgG crystal structure, considering the presumed orientation of the Cys side chain: such residues (Cys side chains may be partially shielded from interaction with another antibody but may still react with the small molecule payload) are more advantageous than residues with greater surface accessibility but with an orientation that enables interaction with large macromolecules such as dimers. Criteria 3) was performed to promote conservative mutations to minimize the destabilizing effect of mutations on the antibody. Likewise, criterion 4) is used to avoid functional changes to the antibody, such as effects on FcRn and protein a binding, which may affect the pharmacokinetic properties of the antibody or may lead to loss of purification process, respectively. The residues excluded based on criterion 4 are listed in table 6. The localization of 88 selected mutation sites in the structural model of hIgG1/κ indicated that the selected sites were surface accessible (FIG. 2).

TABLE 6 surface accessibility of amino acid residues of human IgG1 heavy chain. Surface accessibility was calculated using Surface Racer 5.0 and expressed as square angstroms

"excluded sites" indicate sites excluded from selection for the reasons mentioned in example 1. A "selected site" is a site selected for substitution to Cys in the present invention. />

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Table 7 surface accessibility of amino acid residues of human kappa light chain. Surface accessibility was calculated using Surface Racer 5.0 and expressed as square angstroms

A "selected site" is a site selected for substitution to Cys in the present invention. />

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Example 2 preparation of Trastuzumab Cys mutant antibodies

DNA encoding the variable regions of the heavy and light chains of trastuzumab was chemically synthesized and cloned into two mammalian expression vectors, pogo-HC and pogo-LC, containing the constant regions of human IgG1 and human kappa light chains, yielding two wild-type constructs, pogo-trastuzumab HC and pogo-trastuzumab LC, respectively. In the vector, expression of the antibody heavy and light chain constructs in mammalian cells is driven by a CMV promoter. The vector contains a synthetic 24 amino acid signal sequence at the N-terminus of the heavy or light chain: MKTFILLLLWVLLLWVIFLLPGATA (SEQ ID NO: 99) to direct its secretion from mammalian cells. This signal sequence has been shown to be at the guide 293 Freestyle ^TM Is effective in protein secretion of hundreds of mammalian proteins in cells. Cys mutant constructs were prepared in trastuzumab using oligonucleotide-directed mutagenesis. 88 pairs of mutation primers (Table 8) were chemically synthesized, corresponding to the 88 Cys mutation sites selected in the constant regions of the human IgG1 heavy and kappa light chains described in example 1. The sense and antisense mutation primer pairs were mixed prior to PCR amplification. PCR reactions were performed by using Pfuultra II Fusion HS DNA polymerase (Stratagene) using pOG-trastuzumab HC and pOG-trastuzumab LC as templates. After the PCR reaction, the PCR products were verified on agarose gels and treated with DPN I, followed by transformation in DH5a cells (Klock et al, (2009) Methods Mol biol.498: 91-103).

The sequence of the 88 Cys mutant constructs was verified by DNA sequencing. The full-length amino acid sequence of the wild-type trastuzumab heavy chain is shown as SEQ ID NO 1, and the full-length amino acid sequence of the light chain is shown as SEQ ID NO 90. The protein sequences encoding the constant regions of 59 trastuzumab HC Cys mutant constructs (SEQ ID NO:2-SEQ ID NO: 60) and 29 trastuzumab LC Cys mutant constructs (SEQ ID NO:61-SEQ ID NO: 89) are shown in Table 9 and Table 10, respectively. Amino acid residues in human IgG1 heavy chain and human kappa light chain are numbered by the Eu numbering system (Edelman et al, (1969) Proc Natl Acad Sci U S A, 63.

TABLE 8 DNA sequences of mutation primers used to prepare the 88 Cys mutant heavy and light chains of human IgG 1.

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TABLE 9 amino acid sequence of the constant region of the Cys mutant construct in human IgG1 heavy chain. SEQ ID NO:1 is the sequence of full-length trastuzumab (human IgG 1). SEQ ID NO 2-SEQ ID NO 60 show the sequence ID number of the 59 Cys mutant construct in the heavy chain of human IgG1, showing only the sequence of the constant region.

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Table 10.29 amino acid sequences of constant regions of the human kappa light chain Cys mutant constructs. SEQ ID NO 61 is the sequence of the constant region of the wild-type human kappa light chain. SEQ ID NO:62-SEQ ID NO:90 indicate the sequence ID number of the 29 Cys mutant construct in the constant region of human kappa light chain.

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Example 3 transfer of Trastuzumab heavy and light chain Cys mutations into different antibodies

For trastuzumab, all Cys mutations selected for drug payload attachment were located in the constant regions of their human IgG1 heavy and human kappa light chains. Because the constant regions of antibodies are highly conserved in primary sequence and structure, cys mutant residues identified as good payload attachment sites in the trastuzumab context will also serve as preferred attachment residues in other antibodies. To demonstrate the transferability of these generic conjugation sites to other antibodies, we cloned a panel of Cys mutations into antibody 14090. Antibody 14090 is an antibody with a human IgG1 heavy chain and a human λ light chain that binds to a different target protein than trastuzumab. DNA encoding the variable region of antibody 14090 was cloned into seven selected pieces of the pogo trastuzumab HC Cys mutant plasmid constructs (SEQ ID NOs listed in table 11) to replace the variable region of the trastuzumab construct in the plasmid as described in example 2. As a result, the amino acid sequences of the heavy chain constant regions in the corresponding seven Cys constructs of antibody 14090 and trastuzumab were identical (fig. 3). Subsequent experiments showed that these sites can be easily conjugated. In contrast, due to the high similarity in primary sequence and tertiary structure of different human IgG isotypes (fig. 4), cys mutations on the kappa light chain of trastuzumab can be readily transferred to the equivalent light chain on human antibodies containing heavy chains of different isotypes. Likewise, sites identified in the constant region of IgG1 can be transferred to IgG2, igG3, and IgG 4.

Example 4 cysteine mutations in human lambda light chain

Human λ and κ light chains have little amino acid sequence similarity (fig. 5A). Mutations in the lambda light chain of antibody 14090 were selected based on the approximate similarity of residues in a Protein database structure entry 3g6d.pdb containing Fab of human lambda light chain with reference to the position of the desired residue in the kappa light chain of trastuzumab (fig. 5A and B). Oligonucleotide-directed mutagenesis (Higuchi et al 1988) was used in combination with the PIPE cloning strategy (Klock and Lesley, 2009) to generate seven additional Cys mutant constructs in the antibody 14090- λ light chain plasmid. The mutation primers used to generate Cys point mutations in the lambda light chain are listed in table 12. Secretion of antibody 14090 is also directed by a synthetic 24 amino acid signal sequence: MKTFILLLLWVLLLWVIFLLPGATA (SEQ ID NO: 99). The sequence of the antibody 14090 Cys construct was verified by DNA sequencing. The sequence of the constant region of the human wild-type lambda light chain is shown as SEQ ID NO 91. The protein sequences encoding the seven Cys mutant constructs in the light chain (SEQ ID NO:92-SEQ ID NO: 98) are shown in Table 13. Subsequent experiments will show that these Cys mutants are efficiently conjugated to the ADC payload. Since all these mutants are located in the constant region of the human lambda light chain, these conjugation sites can be easily transferred to other antibodies with lambda light chains.

Table 11 sequence ID number of trastuzumab heavy chain Cys construct used to clone the variable region of antibody 14090.

Sequence ID NO of trastuzumab HC Cys construct:
	SEQ ID NO:5
SEQ ID NO:8
	SEQ ID NO:9
SEQ ID NO:10
	SEQ ID NO:18
SEQ ID NO:48
	SEQ ID NO:50

TABLE 12 nucleotide sequences of primers used to mutagenize seven Cys mutant constructs in the lambda light chain of human IgG 1.

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Table 13 amino acid sequence of the constant region of the Cys mutant construct in the light chain of antibody 14090 λ. 91 is the sequence of the constant region of a wild-type human lambda light chain. SEQ ID NO 91-98 indicate the sequence of the 7 Cys mutants in the constant region of the human lambda light chain of antibody 14090.

Example 5 in 293 Freestyle ^TM Expression and purification of Cys mutant antibodies in cells

The heavy and light chain plasmids were co-transfected at 293 Freestyle using the method previously described (Meissner, et al, biotechnol Bioeng.75:197-203 (2001)) by co-transfection of the heavy and light chain plasmids ^TM A Cys mutant that expresses trastuzumab antibody in a cell. Prepared using Qiagen plasmidKits were prepared and DNA plasmids for co-transfection were prepared according to the manufacturer's protocol. In Freestyle ^TM Expression Medium (Invitrogen) at 37 ℃,5% CO ₂ Under suspension culture of 293 Freestyle ^TM A cell. The day before transfection, the cells were divided into 0.7x10 ⁶ Cells/ml to fresh medium. On the day of transfection, cell densities typically reach 1.5x10 ⁶ Cells/ml. Using a PEI method, using a mixture of 1:1 (Meissner et al, 2001). The transfected cells were then cultured for a further 5 days. The medium was collected from the culture by centrifugation at 2000x g for 20 minutes and filtered through a 0.2 micron filter. Using protein A-Sepharose ^TM (GE Healthcare Life Sciences) the expressed antibody was purified from the filtered medium. From protein A-Sepharose by elution buffer (pH 3.0) ^TM The antibody IgG was eluted from the column and immediately neutralized with 1M Tris-HCl (pH 8.0) followed by buffer exchange to PBS.

Transient transfection of 293 Freestyle ^TM The expression level of the 88 Cys trastuzumab mutant antibodies was similar to that of wild-type trastuzumab with an average yield of 18.6mg/L +/-9.5mg/L (table 14), suggesting that single point mutations in selected sites did not significantly alter retention of expressed antibody by the cell secretion machinery. Analysis of purified trastuzumab Cys mutant antibodies using non-reducing SDS PAGE showed that the Cys mutant antibodies did not form disulfide-linked oligomers through the engineered cysteine (fig. 6). Size exclusion chromatography (figure 7) further supports the conclusion that all Cys mutant trastuzumab antibodies are monomeric. HPLC reverse phase analysis of the mutant antibodies also suggested that most Cys mutant antibodies were indistinguishable from wild-type trastuzumab in terms of retention time and homogeneity (fig. 8). Analysis of non-reductively deglycosylated full-length trastuzumab LC-R108C by mass spectrometry (intact LC-MS) revealed that most antibodies were modified with two cysteines (fig. 9 and table 15). These observations are consistent with previous publications which show that when at 293 freestyle ^TM When expressed in cells, the thiol group of the engineered cysteine in the trastuzumab Cys mutant antibody is modified by cysteine and reacted with any thiol groupThe modification needs to be removed by a reducing reagent prior to conjugation (Chen, et al, mAbs 1, 563-571, 2009).

Using the PEI method (Meissner et al, 2001), HC and LC plasmids can also be co-transfected at 293 freestyle ^TM A Cys mutant of antibody 14090 is expressed in the cell. The expression level of Cys mutants of antibody 14090 was similar to that of wild-type antibody 14090 (table 16).

TABLE 14 at 293 freestyle ^TM Yield of trastuzumab Cys mutant antibody transiently expressed in cells. The yield was measured by UV absorbance at 280nm after protein a purification.

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TABLE 15 from 293 freestyle ^TM Theoretical and observed masses of trastuzumab LC-R108C antibody after purification in cells.

Table 16: at 293 freestyle ^TM Production of transiently expressed antibody 14090 Cys mutant in cells.

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Example 6 reduction, reoxidation and conjugation of Cys mutant antibodies to MC-MMAF

Because the engineered Cys in antibodies expressed in mammalian cells is modified during their biosynthesis by adducts (disulfides), such as Glutathione (GSH) and/or cysteine, the modified Cys in the initially expressed product is not reacted with thiol-reactive reagents, such as maleimide or bromo-or iodo-acetamide groups. To conjugate the engineered cysteines after expression, glutathione or cysteine adducts need to be removed by reduction of these disulfides, which typically requires reduction of all disulfides in the expressed protein. This can be accomplished by first exposing the antibody to a reducing agent, such as Dithiothreitol (DTT), and then by allowing all of the native disulfide bonds of the antibody to reoxidize to restore and/or stabilize functional antibody structure. Thus, to reduce all disulfide bonds bound between the native disulfide bonds and the GSH adducts of cysteine or engineered cysteine residues, freshly prepared DTT was added to the previously purified trastuzumab and Cys mutant of antibody 14090 to a final concentration of 20 mM. After 1 hour incubation of antibody with DTT at 37 ℃, the mixture was dialyzed against PBS at 4 ℃ for 3 days, with daily buffer changes to remove DTT and re-oxidize the native disulfide bonds. An alternative method is to remove the reducing agent by Sephadex G-25 column desalting. Once the protein was completely reduced, 1mM oxidized ascorbic acid (dehydroascorbic acid) was added to the desalted sample and reoxidation incubation was performed for 20 hours. Both methods produced similar results. However, attempts to follow the reoxidation scheme described in the previous literature using CuSO ₄ Resulting in precipitation of the protein. All examples herein used the above dialysis protocol. Reoxidation restores the intrachain disulfide bonds, while dialysis allows cysteine and glutathione to be attached to newly produced cysteine to be dialyzed away.

After reoxidation, the antibody is ready for conjugation. To the reoxidized antibody in PBS buffer (ph 7.2) was added maleimide-MMAF (MC-MMAF, 10 equivalents relative to antibody, fig. 10). Incubation was performed for 1 to 24 hours. The conjugation process was monitored by reverse phase HPLC, which was able to separate the conjugated antibody from the unconjugated antibody. The conjugation reaction mixture was analyzed on a PRLP-S4000A column (50mm. Times.2.1 mm, agilent) heated to 80 ℃ and elution of the column was performed at a flow rate of 1.5 ml/min by a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA. Elution of proteins from the column was monitored at 280nm, 254nm and 215 nm. A reverse phase HPLC trace of a typical conjugate mixture is shown in fig. 11.

When the conjugation mixture was analyzed by reverse phase HPLC, many Cys sites gave rise to homogeneous conjugation products, as suggested by the uniform monomodal elution profile (fig. 11), while some Cys sites gave rise to heterogeneous conjugation products (fig. 12). The methods described above involve the reduction and reoxidation of the native disulfide bond and the reduction of the bond between the cysteine and GSH adduct of the engineered cysteine residue. During reoxidation, engineered cysteine residues may interfere with the reformation of the correct native disulfide bond through the process of disulfide shuttling. This may result in the formation of mispaired disulfide bonds between engineered cysteine and native cysteine residues or between incorrectly paired native disulfide bonds. Such mispaired disulfide bonds may affect retention of antibodies on reverse phase HPLC columns. The process of mismatch may also result in unpaired cysteine residues other than the desired engineered cysteine. Attachment of maleimide-MMAF to different positions of the antibody affects retention time differently (see discussion below for homologously conjugated ADCs). Furthermore, in addition to the desired conjugation to the engineered cysteine residue, incomplete re-oxidation will leave an antibody with the native cysteine residue, which will react with maleimide-MMAF. Any process that prevents the correct and complete formation of the native disulfide bond after conjugation with maleimide-MMAF will generate a complex HPLC profile (fig. 11). The yield of uniform ADC as measured by UV absorbance of the unpurified reaction mixture varied according to Cys mutation (table 17). Using the reduction/reoxidation protocol and conjugation method described above, 65 of the 88 Cys mutant trastuzumab antibodies yielded homogeneous conjugation products and these sites were the favored sites for Cys substitutions to be made when preparing cysteine engineered antibodies for conjugation.

These 65 Cys-MMAF ADCs were analyzed in detail in various assays: differential Scanning Fluorescence (DSF) was used to measure thermal stability. Analytical size exclusion chromatography (AnSEC) was used to measure aggregation. In vitro antigen-dependent cell killing efficacy was measured by cell viability assay and pharmacokinetic behavior was measured in mice. These assays and the respective results are described in more detail below.

To assess the aggregation status of trastuzumab Cys-MMAF ADCs, the ADCs were analyzed on a size exclusion chromatography column (GE, superdex200, 3.2/30) at a flow rate of 0.1 ml/min in PBS. All 65 Cys-MMAF ADCs were monomeric. Most ADCs contained less than 10% oligomers (fig. 13, table 18), indicating that conjugation of MC-MMAF to trastuzumab Cys mutant constructs at selected sites did not cause aggregation of the antibody.

Table 17 production of MMAF ADCs using trastuzumab Cys mutant constructs. "Hetero" indicates a heterogeneous mixture of species with different retention times in reversed phase HPLC.

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Table 18. Percentage of oligomers in trastuzumab Cys-MMAF ADC formulations as determined by analytical size exclusion chromatography.

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b.d. below the detection limit.

Example 7 in vitro thermal stability assay for Trastuzumab Cys-MMAF ADCs

Conjugation of MMAF payload to trastuzumab can stabilize or destabilize the antibody, resulting in a change in melting temperature of the antibody, which can be determined by Differential Scanning Fluorescence (DSF) based on temperature-induced denaturation monitored by environmentally sensitive dyes, such as sypro orange. ADC samples were aliquoted in triplicate into PBS in 384-well plates (6.7 mM sodium phosphate pH7.2;150mM NaCl). In each well, 8. Mu.l of 0.25mg/ml antibody was mixed with 2. Mu.l of 25x sypro orange dye (Invitrogen). The plates were sealed and analyzed in a Roche LightCycler 480, which recorded 20 fluorescence scans per degree Celsius at temperatures ramping from 30 to 85 ℃. The melting temperature was determined from the first derivative of the fluorescence intensity versus time curve.

A typical thermal transfer assay for wild-type trastuzumab revealed two melting transitions (Tm), tm1 at 69.7 ℃ and Tm2 at 81.2 ℃ (table 19). When trastuzumab Cys-MMAF ADCs were subjected to protein thermostability assays, it was evident that the conjugation of MC-MMAF to the antibody induced different Tm changes depending on the site of conjugation (table 19). When MC-MMAF is conjugated to most Cys sites in the CH1 or CH3 domain, the resulting ADCs, e.g., HC-K356C-MMAF, show a pattern similar to wild-type anti-Her with little change in Tm1 and Tm 2. However, when MC-MMAF is conjugated to a Cys site located in the CH2 domain, a decrease in Tm1 is observed for most sites, while Tm2 remains largely unchanged. The Tm1 reduction observed for most CH2 domain Cys-MMAF conjugates varied between 5 ℃ to 26 ℃. The two ADCs with the greatest decrease in Tm1 were HC-T335C-MMAF and HC-S337C-MMAF, with Tm1 at 42 ℃ and 45 ℃ respectively (FIG. 14). The results indicate that the location of MC-MMAF conjugation can have a significant effect on the stability of ADCs.

TABLE 19 melting temperatures Tm1 and Tm2 of trastuzumab Cys-MMAF ADCs as observed by Differential Scanning Fluorometry (DSF).

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n.d. not determined, n.a. is not applicable as a broad shift in Tm2 prevents accurate Tm determination

Example 8 cell proliferation assay to measure in vitro cell killing efficacy of Cys ADCs

Cells that naturally express the target antigen or cell lines engineered to express the target antigen are often used to determine the activity and efficacy of the ADCs. To evaluate the cell killing efficacy of trastuzumab ADCs in vitro, two engineered cell lines, MDA-MB231 clone 16 and clone 40, and HCC1954 cells (Clinchy B, gazdar a, rabinovsky R, yeffenof E, gordon B, vitetta es. Breast Cancer Res treat. (2000) 61. MDA-MB231 clone 16 cells stably expressed high copy number (-5X 10) ⁵ Copies/cell) whereas clone 40 expressed low copy numbers (-5 x 10) ⁵ Copies/cell) of human Her2.HCC1954 cells endogenously express high levels (-5 x 10) on the surface ⁵ Copies/cell) of human Her2. To determine the cell killing efficacy of antibody 14090 ADCs, CMK11-5 and Jurkat cells were used. Although CMK11-5 cells expressed high levels of antigen against antibody 14090 on the cell surface, there was no detectable antigen expression in Jurkat cells. The antigen-dependent cytotoxic effect should only kill cells expressing sufficient antigen on the cell surface, not cells lacking the antigen. Cell-Titer-Glo was used 5 days after incubation of cells with various concentrations of ADCs ^TM (Promega) cell proliferation assays were performed (Riss et al, (2004) Assay Drug Dev technol.2: 51-62). In some studies, cell-based assays are high throughput and are performed in automated systems (Melnick et al, (2006) Proc Natl Acad Sci U S A.103: 3153-3158).

Trastuzumab Cys-MMAF ADCs killed MDA-MB231 clone 16 and HCC1954, but did not kill MDA-MB231 clone 40 cells, among others (FIG. 15). IC of trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 cell assay ₅₀ Varied between 30pM and 200pM (Table 20, FIG. 16). Class ISimilarly, antibody 14090 Cys-MMAF ADC displayed antigen-dependent cell killing in a cell proliferation assay. Antibody 14090 Cys-MMAF ADCs killed CMK11-5 cells expressing the antigen, but did not kill antigen negative Jurkat cells (fig. 17). IC of antibody 14090-MMAF ADC in CMK11-5 cell proliferation assay ₅₀ Varied between 400pM and 1nM (Table 21).

TABLE 20 MDA-MB231 clone 16Her2 ⁺ IC of trastuzumab Cys-MMAF ADCs in cell proliferation assay ₅₀ 。

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TABLE 21 IC of antibody 14090 Cys-MMAF ADCs in CMK11-5 cell proliferation assay ₅₀ 。

Antibody 14090 Cys-MMAF ADC	IC ₅₀ (M)
		HC-S124C	9.26E-04
HC-T139C	1.22E-03
		HC-E152C	4.60E-04
HC-L174C	6.02E-04
		HC-K360C	8.56E-04
HC-S375C	4.38E-04
		LC-A143C	7.09E-04
LC-A147C	1.14E-03
		LC-V159C	5.41E-04
LC-T163C	6.38E-04
		LC-S168C	1.06E-03

Example 9 pharmacokinetic Studies of Trastuzumab Cys-MMAF ADCs

Long serum half-lives have been shown to be critical for high in vivo efficacy of ADCs (Hamblett et al, "Effects of drug loading on the antibody activity of monoclonal antibody drug conjugate," Clin Cancer Res., 10. Attaching a generally hydrophobic drug payload to an antibody may significantly affect the properties of the antibody, and this may result in rapid clearance of ADCs in vivo (Hamblett et al, 2004) and poor in vivo efficacy. To assess the effect of different conjugation sites on clearance of MMAF ADCs in vivo, pharmacokinetic studies were performed in non-tumor bearing mice using 65 trastuzumab Cys-MMAF ADCs. To detect ADCs containing MMAF in mouse plasma, anti-MMAF antibodies were generated. An ELISA assay for detecting ADCs to capture trastuzumab IgG molecules and anti-human IgG (anti-hIgG) antibodies and MMAF antibodies from plasma was developed using the extracellular domain of human HER2 for signal generation in two separate assays. Two ELISA assays measure serum concentrations of trastuzumab antibody and "intact" ADC, as discussed in more detail below, respectively.

Three mice per group were administered with a single dose of trastuzumab Cys-MMAF ADC at 1 mg/kg. 10 plasma samples were then collected over a two week period and all trastuzumab IgG molecules, including trastuzumab Cys-MMAF ADCs and trastuzumab lacking MMAF, were captured by ELISA assay using the extracellular domain of human HER 2. The anti-MMAF and anti-hIgG antibodies were then used for detection in two separate assays. The anti-MMAF antibody ELISA only measured the concentration of trastuzumab MMAF conjugate and the anti-hIgG ELISA quantified trastuzumab Cys-MMAF conjugate and trastuzumab antibody lacking MMAF. A standard curve was generated for each ADC separately using the same material injected into mice. If no drug loading change of trastuzumab Cys-MMAF ADC occurs after injection into mice, then assays using anti-MMAF and anti-hIgG should therefore yield the same concentration readings. For trastuzumab Cys-MMAF ADCs that lost some MMAF payload, an ELISA assay using anti-MMAF antibodies would measure lower concentrations than anti-hIgG ELISA. Comparison of the two concentration readings thus allows measurement of drug release from trastuzumab Cys-MMAF ADCs during in vivo incubation in mice.

63 of the 65 ADCs exhibited similar pharmacokinetic profiles as the unconjugated wild-type trastuzumab antibody as measured by anti-hIgG ELISA (fig. 18, 19, 20), indicating that MC-MMAF payload conjugation to these sites did not significantly affect antibody clearance. Two exceptions are HC-T335C and HC-S337C. The conjugation of MC-MMAF to these two sites resulted in rapid clearance of ADCs as measured by anti-MMAF and anti-hIgG ELISA (figure 21). Protein heat transfer assays revealed that Tm1 of trastuzumab HC-T335C-MMAF and trastuzumab HC-S337C-MMAF decreased from 69 ℃ to 42 ℃ and 45 ℃ respectively in the wild-type trastuzumab antibody (fig. 14). The conjugation of MC-MMAF to two sites significantly reduced the thermal stability of the ADC (27 ℃ and 24 ℃ respectively). Tm1 changes by less than 8 ℃ for 63 ADCs that showed similar pharmacokinetic profiles to the unconjugated antibody, suggesting that rapid clearance may be associated with low thermostability of the ADCs.

To determine the chemical stability of the linkage between MMAF payload and antibody at multiple Cys sites, the concentration of trastuzumab Cys-MMAF ADC measured by anti-MMAF ELISA and the concentration of all trastuzumab molecules measured by anti-hIgG ELISA were compared to each other for each sample. Within the error of the measurement, many trastuzumab Cys-MMAF ADCs exhibited good overlap between the two concentrations over the two week period, suggesting that the bond between MC-MMAF and cysteine introduced at these sites was stable during the circulation in mice during this period (fig. 18, 19). In contrast, some trastuzumab Cys-MMAF ADCs exhibited significant drug loss as indicated by higher anti-hIgG readings than anti-MMAF readings (fig. 20). For some trastuzumab Cys-MMAF ADCs, the concentration of ADC is about 50% of hIgG. These results suggest significant differences in the stability of the thiol-maleimide bond of drug payloads conjugated to different sites, as has been previously suggested (Shen et al nat. Biotechnol.2012,30 (2): 184-9). Sites with good stability are preferred sites for the preparation of ADCs as described herein.

In pharmacokinetic studies, the area under the plasma concentration versus time curve (AUC) is an important parameter for assessing the overall clearance and bioavailability of administered drugs. In our pharmacokinetic studies, two AUC values, AUC-MMAF and AUC-hIgG, were calculated from measurements using anti-MMAF and anti-hIgG ELISA, respectively, for each trastuzumab Cys-MMAF ADC. The ratio of AUC-MMAF to AUC-hIgG varied between 0.4 and 1.2 for all trastuzumab Cys-MMAF ADCs (Table 20). Figures 18, 19 and 20 include PK curves for ADCs over the full range of observed AUC-MMAF/AUC-hIgG ratios and demonstrate variability and uncertainty in the measurements. Ratios of AUC-MMAF to AUC-hIgG >1 (table 20) suggest uncertainties >25% because the ratio should remain close to 1 if no drug loss occurs. As shown in table 20, of the 63 trastuzumab Cys-MMAF ADCs with measurable AUCs from two ELISAs, 40 ADCs exhibited ratios of AUC-MMAF/AUC-hIgG >0.7, indicating that within the accuracy of the measurement, little MMAF drug loss was observed at these sites after administration in mice. However, 23 ADCs exhibited ratios of AUC-MMAF/AUC-hIgG <0.7, suggesting that the amount of MMAF payload conjugate at these 23 sites decreased significantly during in vivo incubation in mice.

Differences in the stability of maleimide attachment at different conjugation sites have previously been reported for Cys engineered ADCs (see discussion and reference to Shen et al, (2012) Nat Biotechnol.22;30 (2): 184-9). For preferred sites exhibiting enhanced serum stability, the antibody environment is likely to catalyze the hydrolysis of the succinimide ring formed by the reaction of maleimide with cysteine. The hydrolyzed form cannot revert and release the maleimide drug. As such, the ability of the antibody environment to catalyze ring hydrolysis cannot be predicted and is an unexpected property of certain engineered Cys sites. Based on this criterion, sites in table 22 having a ratio AUC (MMAF)/AUC (hIgG) of greater than 0.7 are thus particularly suitable sites for cysteine substitution, and, where applicable, sites having a ratio of about 0.9 or higher than 0.9 are particularly preferred cysteine substitution sites for the purposes of the present invention. These include heavy chain positions 322, 334, 121, 288, 171, 139, 360, 117, 392, 375, 292, 333, 174, 258, 337, 422, 320, 390, and 335; and light chain positions 107, 203, 108 and 114.

TABLE 22 AUC-MMAF and AUC-hIgG of trastuzumab Cys-MMAF ADCs in mice

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n.a. not applicable.

Example 10: combining Cys sites to generate antibody drug conjugates with drug-to-antibody ratios above 2

Antibody conjugates produced by conjugation to lysine residues or partially reduced native disulfide bonds are often characterized by a drug-to-antibody ratio (DAR) of between 3 and 4. For certain indications it may be desirable to generate ADCs with higher DAR, which in principle can be achieved by introducing multiple Cys mutations in the antibody. As the number of Cys mutations increases, the probability that the mutations will interfere with the required re-oxidation process during ADC preparation increases, thus resulting in an increase in heterogeneous products. In this study, a large number of single-site heavy and light chain Cys mutants with good reoxidation behavior were identified.

To demonstrate that several conjugation sites can be combined for generating ADCs with DAR higher than 2, 293 Freestyle described in example 5 ^TM Several preferred single-site Cys constructs co-expressing trastuzumab with the light and heavy chains of antibody 14090 (table 23) in cells. Purified antibodies, each containing one Cys mutation in the heavy chain and one Cys mutation in the light chain, were reduced and re-oxidized and conjugated to MC-MMAF as described in example 6. Reverse phase high pressure liquid chromatography demonstrated a single well-defined elution peak, suggesting efficient re-oxidation of the native disulfide bond. Reverse phase high pressure liquid chromatography after MC-MMAF conjugation also showed mainly a single elution peak for the DAR 4 ADC species. DAR was confirmed to be 4 for all ADCs in table 23 by mass spectrometry. Production yields varied between 16-24mg/L transient cell cultures. ADCs are predominantly monomeric as determined by analytical size exclusion chromatography; only a small amount of aggregation could be detected for 2 out of 8 antibodies (table 23). Musical composition Tocuzumab and 14090ADCs exhibited antigen-dependent cell killing in the MDA-MB231 clone 16 and CMK1105 cell proliferation assay, respectively (table 23).

TABLE 23 Properties of Cys engineered MMAF ADCs with DAR of 4

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n.d. is not detectable,

no effect: no evidence of cell killing at the highest concentration (66 nM) evaluated

SEQ ID NOs only specify the constant regions of the antibody sequence.

Example 11 Cys site selection based on ADC hydrophobicity

To further optimize the selection of Cys mutants and mutant combinations for the preparation of ADCs with

DAR

2, 4, 6 and 8, the properties of MMAF ADCs prepared with single-site trastuzumab Cys and pci mutants were analyzed (preparation of pci ADCs is described in patent application 55573), and the accessibility of the conjugation site and solvent exposure were examined in the crystal structure of IgG.

One of the most informative data was the observation that trastuzumab pci-MMAF ADCs varied greatly in hydrophobicity when payloads were attached to different sites (fig. 23). By Hydrophobic Interaction Chromatography (HIC) using a TSKgel Phenyl-5PW column (Tosoh Bioscience, TSKgel Phenyl-5PW,13 μ M,21X150mM, stainless steel, cat #07656; electrophoresis buffer A: 1.5M ammonium sulfate in 20mM NaPi (pH 7.4); buffer B: 20% isopropanol in 20mM NaPi (pH 7.4); flow rate 5 ml/min; linear gradient of 20% -80% buffer B over 90 min; monitored by UV absorbance at 280 nm). Surprisingly, although the only difference was the site of ABA-MMAF attachment (fig. 23), the retention time of DAR 2 species was observed to vary greatly between ADCs. HIC separates molecules on the basis of hydrophobicity. All DAR 2 ADCs had a retention time greater than that of the unconjugated antibody (WT =45 min, fig. 23), which was expected when a hydrophobic drug molecule, such as ABA-MMAF, was attached to the antibody. However, attaching payloads at different sites increases the hydrophobicity of the ADC to different degrees.

The surprising large difference in retention time can be theoretically explained from observations of the localization of attachment sites on the antibody structure (fig. 24): if the drug payload is attached to an exposed site outside the antibody, for example at HC-K288Pcl, HC-N286Pcl, HC-V422Pcl, HC-L398Pcl and HC-S415Pcl, the residence time is longer, where the residence time between 87 and 94 minutes is measured for each ADCs (FIG. 23). Conversely, if the payload attaches to an internal site, such as the cavity formed between the variable domain and the CH1 domain of an antibody (e.g., HC-P153Pcl, HC-E152Pcl, HC-L174Pcl, HC-P171Pcl, LC-R142Pcl, LC-E161Pcl, LC-E165Pcl, LC-S159 Pcl) or the large opening between the CH2 and CH3 domains (e.g., HC-K246C, HC-S375Pcl, HC-T393Pcl, HC-K334 Pcl), then the HIC residence time increases to only 47 to 57 minutes, as the payload is partially isolated from interaction with the solvent and the HIC column. For other sites, e.g., LC-K107Pcl and HC-K360Pcl, relative to the exposed sites, intermediate retention times of 70 and 83 minutes were measured.

It is generally believed that reducing the hydrophobicity of the protein drug is beneficial because it may reduce aggregation and clearance from the circulation. We propose that the HIC data presented in figure 23 enables selection of preferred payload attachment sites. Independent of the conjugation chemistry and payload type, it should be beneficial to conjugate the drug payload at a site where the payload is sequestered from interaction with the solvent and attachment minimally increases the hydrophobicity of the antibody. When 4, 6 or 8 drugs are attached to each antibody, or when a particularly hydrophobic payload is used, it may be particularly beneficial to carefully select the attachment site that results in the least change in hydrophobicity.

Cys sites selected for ADCs with low hydrophobicity:

to minimize the hydrophobicity of the ADCs, sites were chosen that would point to the interior of multiple protein domains of the antibody. Where applicable, an assay based on antibody structure and behavior of existing ADCs with DAR =2 (behavior = retention time on HIC and/or delayed retention time on AnSEC with conjugate interacting with SEC resin) was selected. Among the Cys sites identified in tables 1 and 2, the sites listed in table 24 meet the criteria described above.

All ADCs were analyzed by Hydrophobic Interaction Chromatography (HIC). Trastuzumab MMAF ADCs conjugated at exposure sites HC-K360C, LC-K107C, HC-E258C, and HC-R292C were used for comparative purposes. The results are shown in table 25. Trastuzumab Cys-MMAF ADCs and unconjugated wild-type antibody were analyzed on a TSKgel Butyl-NPR column as described above. For comparison, HIC data previously obtained for Pcl-MMAF ADCs on TSKgel Phenyl-5PW are also presented (FIG. 23). Despite the different instrument operations and protocols, and despite some expected variability due to the different geometry and structure of the two linkers, the ratio between retentions of ADCs at the same location but conjugated by different conjugation methods remains nearly constant. HIC data suggest that retention time is indeed a measure of how well the payload is sequestered inside the antibody independent of the attachment chemistry and linker structure. As expected, the relative grades of the different attachment sites remained largely the same for Pcl-MMAF and Cys-MMAF ADCs.

HC-E333C, HC-K392C, and HC-K326C, attached to the selected sites in Table 24, produced MMAF ADCs with HIC retention times similar to the exposed sites ADCs LC-K107C-MMAF, HC-E258C-MMAF, HC-R292C-MMAF, and HC-K360C-MMAF (Table 28). Attachment to HC-E152C, LC-E165C, HC-P171C, LC-R142C, LC-E161C, HC-L174C, and HC-S124C sites increased the retention time of the resulting ADC by less than 15% compared to the unconjugated wild-type antibody. These sites were all located in the CH1 domain or on the Light Chain (LC) and HIC residence time data suggested that they are preferred attachment sites. Among the CH3 domain sites, HC-K334C and HC-S375C exhibit minimal increase in hydrophobicity upon conjugation, making them preferred attachment sites.

TABLE 24 Cys mutant sites

Table 25 Hydrophobic Interaction Chromatography (HIC) retention time of DAR 2 species for trastuzumab MMAF ADCs.

Comparing Cys and pci conjugation chemistries, the two groups coincided well: hiding sites for drugs conjugated by one chemistry also tends to hide drugs when conjugated by another chemistry. Some variability is expected due to the different geometries of the two joint systems.

^a Analytical HIC: tosoh Bioscience (King of Prussia, PA, USA) TSKgel Butyl-NPR column (100 mM. Times.4.6 mM,2.5 μ M), running buffer A50 mM sodium phosphate, 1.5M ammonium sulfate, pH 7.0; buffer B50 mM sodium phosphate, pH 7.0; the gradient consists of a linear gradient of 20-100% B within 100% A for 5 minutes, followed by 40 minutes; monitoring was by UV absorbance at 280 nm.

^b Semi-preparative HIC: tosoh Bioscience (King of Prussia, PA, USA), TSKgel Phenyl-5PW,13 μm,21X150mm; running buffer A1.5M ammonium sulfate in 20mM NaPi (pH7.4); buffer B20% isopropanol in 20mM NaPi (pH 7.4); flow rate 5 ml/min; a linear gradient from 20% to 80% buffer B over 90 min; monitoring was by UV absorbance at 280 nm.

Detailed analytical HIC protocol:

analytical HIC data for trastuzumab Cys-MMAF ADCs were collected using a Tosoh Bioscience (King of Prussia, PA, USA) TSKgel Butyl-NPR column (100 mm. Times.4.6 mm,2.5 μm) mounted on a Dionex UltiMate 3000 HPLC (Sunnyvale, calif., USA). The method consisted of a binary gradient of buffer A (50 mM sodium phosphate, 1.5M ammonium sulfate, pH 7.0) and buffer B (50 mM sodium phosphate, pH 7.0). Samples were prepared by diluting approximately 50 μ g of antibody (PBS) with an equal volume of 3M ammonium sulfate. The gradient consisted of a linear gradient of 20-100% a at 100% for 5 minutes, followed by 40 minutes, and finally re-equilibration for 10 minutes at initial conditions before the next injection. The separation was monitored by UV absorbance at 280 nm.

Preparation and characterization of

DAR

4, 6 and 8 Cys ADCs

Cys mutations can be combined for the preparation of

DAR

4, 6 and 8 ADCs. In general, the preferred combination is a combination of two Cys mutations, resulting in ADCs with DAR 4. Some examples involving combining Heavy Chain (HC) Cys mutants with Light Chain (LC) Cys mutants for making DAR 4 ADCs are shown for trastuzumab and antibody 14090 in example 10. Additional data is provided in table 26. Based on HIC data and examination of attachment sites in IgG crystal structures, additional Cys combinations were prepared using the protocols described in examples 2, 5 and 6. Data for selected examples of MMAF ADCs are shown in table 26. In addition, selected heavy chain sites were combined and the double Cys mutation of the heavy chain was cloned following the protocol set out in example 2. Antibodies characterized by two HC Cys mutations were prepared and conjugated following the protocols described in examples 5 and 6.

To prepare DAR 4 ADCs, the combinations included single-site mutations listed in table 24. The combination of single sites produced ADCs with low hydrophobicity (table 25). In some combinations, one Cys mutation is located in the CH1 domain or on the Light Chain (LC), and the second site is located in the CH3 domain. Examples of such combinations are HC-E152C and HC-S375C, with LC-E165C and HC-S375C, with HC-E152C and HC-K334C, with the characteristic Cys mutant antibodies of LC-E165C and HC-K334C.

ADCs with DAR 6 and 8 can also be prepared when combining three or four Cys mutations in one antibody. The selected heavy chain combinations were combined for

DAR

4, 6 and 8 ADCs preparation. The double and triple Cys mutations of the heavy chain were cloned according to the protocol outlined in example 2. Antibodies characterized by two, three and four Cys mutations were prepared and conjugated according to the protocols described in examples 5 and 6. The characteristics of some DAR 4, DAR 6 and DAR 8 ADC examples are summarized in table 26. Some of these ADCs had surprisingly good PK properties as shown in figure 25. Antibody 14090 is mouse cross-reactive, and thus, as expected, antibody 14090 ADCs cleared more rapidly than trastuzumab ADCs that did not bind any mouse antigen.

Combinations include those with three and four single-site mutations listed in table 24. Combinations include those sites that produce ADCs with low hydrophobicity (table 25). The combination includes one Cys mutation localized in the CH1 domain or on the Light Chain (LC), and optionally an additional one to three sites located in the CH3 domain. Examples of such combinations include antibodies characterized as being a combination of HC-E152C or LC-E165C, with HC-S375C, with the Cys mutation of HC-K334C and/or HC-K392C. Preferred combinations for the preparation of DAR 6 and DAR 8 ADCs are shown in tables 27 and 28, respectively.

With some exceptions, attachment of MMAF at all Cys sites studied resulted in ADCs with high thermal stability (example 7, table 19), propensity for oligomerization (example 6, table 18), and good pharmacokinetic properties of DAR 2 ADCs (example 9, table 22, figure 18). When relatively soluble payloads such as MMAF are used, differences in ADC hydrophobicity are clearly not translated into large differences in biophysical and pharmacokinetic properties. Indeed, as indicated above, it is even possible to use exposed, "hydrophobic" sites, such as HC-K360C, in combination with more preferred attachment sites to prepare DAR 4, DAR 6 and DAR 8 MMAF ADCs with acceptable pharmacokinetic properties. However, when not functioning well, with more hydrophobic payloads, careful selection of the attachment site that results in minimal change in hydrophobicity may be critical to allow for the preparation of non-aggregated ADCs with good pharmacokinetic properties. For such hydrophobic payloads, it may be beneficial to use a combination of sites that reduces the increase in hydrophobicity when 4, 6 or 8 drugs are attached per antibody.

TABLE 26 characterization of selected

DAR

4, 6, and 8 MMAF ADCs prepared using Cys mutation combinations.

* AUC calculations based on mouse PK measurements using anti-MMAF and anti-IgG ELISA assays.

n.d.; not detected, below the limit of quantitation.

TABLE 27 preferred Cys site combinations for the preparation of DAR 6 ADCs.

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TABLE 28 preferred Cys site combinations for the preparation of DAR 8 ADCs.

ADC combination	Site	1	Site 2	Position 3	Position 4
						1	HC-E152C	HC-S375C	HC-K334C	HC-K392C
2	HC-E152C	HC-S375C	HC-E333C	HC-K392C
					3	LC-E165C	HC-S375C	HC-K334C	HC-K392C
4	LC-E165C	HC-S375C	HC-E333C	HC-K392C

Example 12 in vivo potency studies of trastuzumab Cys-MMAF ADCs.

In vivo xenograft tumor models mimicking biological activity were observed by transplanting related and well characterized human primary tumors or tumor cell lines into immunodeficient nude mice. Studies using anticancer agents to treat tumor xenograft mice have provided valuable information about the in vivo efficacy of test agents (Sausville and Burger, 2006). Because MDA-MB231 clone 16 cells were sensitive to trastuzumab Cys-MMAF ADCs in an antigen-dependent manner (fig. 15), the cell line was selected as an in vivo model to evaluate trastuzumab Cys-MMAF ADCs. According to the Guide for the Care and Use of Laboratory Animals (NIH publication; national Academy Press, 8) ^th edition, 2001) were performed for all animal studies. MDA-MB231 clone 16 cells were implanted subcutaneously into nu/nu mice (Morton and Houghton, 2007). The size of the tumor reaches 200mm ³ Thereafter, trastuzumab Cys-MMAF ADCs were administered to mice by IV injection at a single dose of 3 mg/kg. Tumor growth was measured weekly after ADC injection. Each treatment group included 9 mice. An example of an in vivo efficacy study using three trastuzumab Cys-MMAF ADCs is indicated in figure 22. Treatment of mice with 3mg/kg trastuzumab Cys-MMAF ADCs caused tumor regression for all three Cys-MMAF ADCs tested (figure 22). No weight loss associated with ADC treatment was observed. The results demonstrate that trastuzumab Cys-MMAF ADCs effectively caused regression of MDA-MB231 clone 16 tumors with a single dose treatment of 3 mg/kg.

Claims

2. The immunoconjugate of claim 1, wherein the substitution of one or more amino acids with cysteine is selected from positions 121, 124, 152, 258, 334, 360, and 392.

3. The immunoconjugate of claim 1 or 2, wherein said antibody or antibody fragment comprises a sequence selected from SEQ ID NOs 4, 5, 10, 17, 18, 29, 35, 42, 43, 48, 50, 54, 290, 291, 292, 293, 294, and 295.

5. The immunoconjugate of claim 4, wherein the substitution of one or more amino acids with cysteine is selected from positions 145 or 165.

6. The immunoconjugate of claim 4, wherein said antibody or antibody fragment comprises a sequence selected from SEQ ID NOs:61, 62, 69, 71, 75, 76, and 77.

7. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region at a position selected from positions 143, 147, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human λ light chain.

8. The immunoconjugate of claim 7, wherein said antibody or antibody fragment comprises a sequence selected from SEQ ID NOs:92, 94, 96, 97, and 98.

9. The immunoconjugate of claim 1, 2 or 3, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with cysteine on its constant region at a position selected from positions 107, 108, 142, 145, 159, 161, and 165 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

10. The immunoconjugate of claim 1, 2 or 3, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with cysteine on its constant region at positions 143, 147, 159, 163, and 168 of a light chain selected from said antibody or antibody fragment, wherein said light chain positions are numbered according to the Kabat system, and wherein said light chain is a human kappa light chain.

11. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of cysteine to two or more amino acid substitutions at positions 152 and 375, or at positions 327 and 375, on a heavy chain constant region, wherein said positions are numbered according to the EU system.

12. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of substitution of two or more amino acids with cysteine at position 107 of the light chain and 360 of the heavy chain on a constant region, wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

13. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400, or 422 of the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

14. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of the light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

15. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine at a position selected from positions 143, 145, 147, 156, 159, 163, and 168 on its constant region of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

16. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of cysteine substitutions of two or more amino acids on its constant region, wherein said combination comprises a substitution at position 375 of an antibody heavy chain and position 165 of an antibody light chain, or at position 334 of an antibody heavy chain and position 165 of an antibody light chain, and wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

17. The immunoconjugate of any one of claims 11, 12 and 16, wherein said drug-to-antibody ratio is about 4.

18. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of three or more amino acids with cysteine on its constant region, wherein said combination comprises a substitution selected from

19. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of three or more amino acids with cysteine on its constant region, wherein said combination comprises a substitution selected from

a. Positions 152, 375 and 392 of the heavy chain of the antibody,

b. positions 152, 334 and 375 of the heavy chain of the antibody, and

c. substitutions at positions 152, 334 and 392 of the antibody heavy chain,

and the positions are numbered according to the EU system.

20. The immunoconjugate of claim 18 or 19, wherein said drug-to-antibody ratio is about 6.

21. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of four or more amino acids with cysteine on its constant region, wherein said combination comprises substitutions at positions 334, 375, and 392 of an antibody heavy chain and position 165 of an antibody light chain, or positions 333, 375, and 392 of an antibody heavy chain and position 165 of an antibody light chain, and wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.

23. The immunoconjugate of claim 21 or 22, wherein said drug-to-antibody ratio is about 8.

24. The immunoconjugate of any one of claims 1 to 23, further comprising a drug moiety.

25. The immunoconjugate of claim 24, wherein the drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker.

26. The immunoconjugate of claim 25, wherein said drug moiety is linked to said sulfur of said cysteine through a cleavable or non-cleavable linker.

27. The immunoconjugate of claim 25, wherein said drug moiety is attached to said sulfur of said cysteine through a non-cleavable linker.

28. The immunoconjugate of claim 25, wherein said immunoconjugate comprises a thiol-maleimide linkage.

29. The immunoconjugate of claim 25, wherein said immunoconjugate comprises S-CH ₂ -C (= O) -linkage or disulfide bond.

30. The immunoconjugate of any one of claims 25-29, wherein said drug moiety is a cytotoxic agent.

31. The immunoconjugate of claim 30, wherein said drug moiety is selected from the group consisting of taxanes, DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, and maytansinoids.

32. The immunoconjugate of any one of claims 1-31, wherein said antibody is a monoclonal antibody.

33. The immunoconjugate of any one of claims 1-31, wherein said antibody is a chimeric antibody.

34. The immunoconjugate of claim 31, wherein said antibody is a humanized or fully human antibody.

35. The immunoconjugate of claim 31, wherein said antibody is a bispecific or multispecific antibody.

36. The immunoconjugate of any one of claims 1 to 32, wherein said antibody or antibody fragment specifically binds to a cell surface marker characteristic of a tumor.

37. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1-36.

38. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine in its constant region selected from position 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 or 422 of the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

39. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine on its constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199 and 203 of the light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain.

40. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine on its constant region selected from positions 143, 145, 147, 156, 159, 163, 168 on the light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

41. The modified antibody or antibody fragment of claim 38, wherein the substitution is at least one cysteine selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 360 and 375 of the heavy chain, and wherein the positions are numbered according to the EU system.

42. The modified antibody or antibody fragment of claim 38, wherein the substitutions are two to six cysteines, wherein the cysteines are at positions selected from 121, 124, 152, 171, 174, 258, 292, 333, 360, and 375 of the heavy chain, and wherein the positions are numbered according to the EU system.

43. The modified antibody or antibody fragment of claim 39, wherein the substitution is at least one cysteine selected from positions 107, 108, 142, 145, 159, 161, and 165 of the light chain, wherein the positions are numbered according to the EU system, and wherein the light chain is a human kappa light chain.

44. The modified antibody or antibody fragment of claim 39, wherein the substitutions are two to six cysteines, wherein the cysteines are at positions selected from the group consisting of 107, 108, 142, 145, 159, 161, and 165 of the light chain, wherein the positions are numbered according to the EU system, and wherein the light chain is a human kappa light chain.

45. The modified antibody or antibody fragment of claim 40, wherein the substitution is at least one cysteine selected from positions 143, 147, 159, 163 and 168 of the light chain, wherein said positions are numbered according to the Kabat system, and wherein the light chain is a human λ light chain.

46. The modified antibody or antibody fragment of claim 38, wherein the substitution is two to six cysteines, wherein the cysteines are at positions selected from 143, 147, 159, 163, and 168 of the light chain, wherein the positions are numbered according to the Kabat system, and wherein the light chain is a human lambda light chain.

47. The modified antibody or antibody fragment of any one of claims 11, 12, 14-22, 38-47, further attached to a drug moiety, and wherein said drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker.

48. The modified antibody or antibody fragment of claim 47, wherein the drug moiety is attached to the sulfur of the cysteine through a linker unit.

49. The modified antibody or antibody fragment of any one of claims 38-48, further comprising at least one Pcl or unnatural amino acid substitution or peptide tag for enzyme-mediated conjugation and/or combinations thereof.

50. A nucleic acid encoding the modified antibody or antibody fragment of any one of claims 38-49.

51. A host cell comprising the nucleic acid of claim 50.

52. A method of producing a modified antibody or antibody fragment comprising incubating the host cell of claim 49 under suitable conditions for expression of said antibody or antibody fragment, and isolating said antibody or antibody fragment.

53. A method of selecting amino acids of an antibody suitable for substitution by cysteine to provide a suitable conjugation site, the method comprising:

(1) Identifying suitable surface exposed amino acids in the antibody constant region to provide a set of initial candidate sites;

(3) For each of the expressed antibodies, determining whether the expressed protein is substantially homogeneous after reduction and reoxidation to provide a functional antibody having a free cysteine at the initial candidate site,

(4) For each protein expressed that is substantially homogeneous and functional, conjugating a cysteine at the initial candidate site with a maleimide moiety and determining whether the thiol-maleimide linkage at that site is destabilized;

54. The method of claim 53, further comprising the step of: the melting temperature is determined for each advantageous cysteine substitution site conjugate and any such site is excluded from the set, wherein the cysteine substitution and conjugation results in a melting temperature that is 5 ℃ or more different from the melting temperature of the parent antibody.

55. The method of claim 53 or 54, further comprising generating an antibody or antibody fragment containing a cysteine at the identified one or more substitution sites.

56. A method of producing an immunoconjugate, comprising attaching a Linker Unit (LU) or linker unit-payload combination (-LU-X) to a cysteine residue in an antibody or antibody fragment, wherein the cysteine is located at a cysteine substitution site selected from the group consisting of 121, 124, 152, 171, 174, 258, 292, 333, 360, and 375 of a heavy chain of the antibody or antibody fragment and 107, 108, 142, 145, 159, 161, and 165 of a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system.

57. The method of claim 56, wherein the immunoconjugate is of formula (I):

LU is a joint unit as described herein;

x is a payload or drug moiety;

and n is an integer from 1-16.